U.S. patent application number 16/234453 was filed with the patent office on 2020-03-19 for devices and methods for nucleic acid extraction.
The applicant listed for this patent is Visby Medical, Inc.. Invention is credited to Jennifer Albrecht, Boris Andreyev, Edward Biba, Victor Briones, Ryan Cena, Jesus Ching, Brian Ciopyk, Adam De La Zerda, Jonathan Hong, Helen Huang, Colin Kelly, Adrienne C. Lam, Gregory C. Loney, Keith Moravick, Valeria Revilla, David D. Swenson.
Application Number | 20200086324 16/234453 |
Document ID | / |
Family ID | 60785483 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200086324 |
Kind Code |
A1 |
Swenson; David D. ; et
al. |
March 19, 2020 |
DEVICES AND METHODS FOR NUCLEIC ACID EXTRACTION
Abstract
Disclosed herein are methods and devices for preparing a sample
of nucleic acid molecules from a biological sample. The methods and
devices may perform similarly to or better than standard sample
preparation methods. The nucleic acid molecules prepared using the
methods and devices provided herein may be utilized for downstream
applications, including polymerase chain reaction (PCR).
Inventors: |
Swenson; David D.; (Santa
Clara, CA) ; De La Zerda; Adam; (Palo Alto, CA)
; Loney; Gregory C.; (Los Altos, CA) ; Revilla;
Valeria; (East Palo Alto, CA) ; Briones; Victor;
(Gilroy, CA) ; Andreyev; Boris; (Foster City,
CA) ; Lam; Adrienne C.; (Fremont, CA) ;
Ciopyk; Brian; (Pleasanton, CA) ; Huang; Helen;
(San Pablo, CA) ; Moravick; Keith; (Mountain View,
CA) ; Kelly; Colin; (San Francisco, CA) ;
Ching; Jesus; (Saratoga, CA) ; Albrecht;
Jennifer; (Sunnyvale, CA) ; Cena; Ryan; (San
Jose, CA) ; Biba; Edward; (Santa Clara, CA) ;
Hong; Jonathan; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Visby Medical, Inc. |
San Jose |
CA |
US |
|
|
Family ID: |
60785483 |
Appl. No.: |
16/234453 |
Filed: |
December 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2017/040112 |
Jun 29, 2017 |
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16234453 |
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62357306 |
Jun 30, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0645 20130101;
B01L 7/525 20130101; B01L 2300/0883 20130101; B01L 2300/0816
20130101; C12Q 1/6844 20130101; B01L 2200/0684 20130101; C12N 15/10
20130101; C12N 9/1276 20130101; B01L 2200/027 20130101; G01N 21/78
20130101; B01L 3/502761 20130101; B01L 7/52 20130101; B01L
2300/1822 20130101; B01L 2200/10 20130101; B01L 2400/0622 20130101;
G01N 2030/8818 20130101; C12Q 1/6806 20130101; B01L 3/502715
20130101; G01N 21/29 20130101; G01N 30/88 20130101; B01L 3/502769
20130101; B01L 2300/1827 20130101; B01L 2400/0644 20130101 |
International
Class: |
B01L 7/00 20060101
B01L007/00; B01L 3/00 20060101 B01L003/00; C12Q 1/6806 20060101
C12Q001/6806; C12Q 1/6844 20060101 C12Q001/6844 |
Claims
1.-21. (canceled)
22. An apparatus, comprising: a housing; a reverse transcription
module disposed within the housing and configured to receive a
sample, the reverse transcription module including a first flow
member and a first heater, the first flow member defining an
reverse transcription flow path having an inlet portion configured
to receive the sample, the first heater fixedly coupled to the
first flow member such that the first heater and the reverse
transcription flow path intersect at multiple locations, the
reverse transcription module configured to perform a reverse
transcriptase reaction on the sample; an amplification module
disposed within the housing and configured to receive an output
from the reverse transcription module, the amplification module
including a second flow member and a second heater, the second flow
member defining an amplification volume, the second heater coupled
to the second flow member, the amplification module configured
amplify the output from the reverse transcription module to produce
a target amplicon; and a detection module disposed within the
housing and configured to receive an output from the amplification
module, the detection module configured to react a reagent with the
target amplicon to produce a signal indicating a presence of the
target amplicon.
23. The apparatus of claim 22, further comprising: a reagent stored
within the housing, the reagent formulated to produce the signal
that indicates the presence of the target amplicon in the sample,
the signal being a colorimetric signal.
24. The apparatus of claim 23, further comprising: a reagent
actuator configured to convey the the reagent from a sealed reagent
container into a holding chamber fluidically coupled to the
detection module when the reagent actuator is moved from a first
position to a second position, the reagent actuator including a
locking shoulder configured to matingly engage a portion of the
housing to maintain the reagent actuator in the second
position.
25. (canceled)
26. The apparatus of claim 22, wherein the detection module is
fixedly coupled within the housing and includes a detection surface
from which the signal that indicates presence of the target
amplicon in the input sample is produced, the detection surface
visible via a detection opening defined by the housing.
27.-28. (canceled)
29. The apparatus of claim 22, further comprising: a power source
disposed within the housing and configured to supply power to the
amplification module, the power source having a capacity of less
than about 1200 mAh.
30.-46. (canceled)
47. The molecular diagnostic test device of claim 22, wherein the
reverse transcription module contains a reverse transcriptase
enzyme and reagents required for the reverse transcriptase
reaction, the reverse transcriptase enzyme and reagents being
present as a lyophilized pellet.
48.-51. (canceled)
52. A method for DNA preparation, comprising: (a) obtaining a
biological sample comprising one or more biological entities,
wherein the biological entities comprise RNA; (b) lysing said one
or more biological entities, thereby releasing a plurality of RNA
molecules therefrom; and (c) performing a reverse transcriptase
reaction on the released RNA molecules to produce a plurality of
DNA molecules, wherein said method extracts said nucleic acid
molecules from said one or more biological entities within 5
minutes or less at a quality sufficient to successfully perform a
polymerase chain reaction (PCR).
53. The method of claim 52, wherein the method is performed by a
handheld device.
54. The method of claim 52, wherein a quality sufficient to
successfully perform a polymerase chain reaction comprises nucleic
acid molecules which amplify with at least 70% efficiency as
determined by a qPCR standard curve.
55. The method of claim 52, wherein the method produces at least
100 .mu.I, of a solution containing the nucleic acid molecules.
56.-64. (canceled)
65. A method of DNA preparation, comprising: conveying a biological
sample comprising RNA into a sample input module of a molecular
diagnostic test device; and actuating the molecular diagnostic test
device to: convey the biological sample to a reverse transcription
module within the molecular diagnostic test device, the reverse
transcription module including a heater and defining a first
reaction volume and a second reaction volume, and further
comprising lyophilized reagents for a reverse transcription
reaction; maintain an input solution containing the biological
sample and the reagents for reverse transcription within the first
reaction volume to reverse transcribe at least a portion of the
biological sample thereby producing a plurality of complementary
DNA (cDNA) molecules; activate the heater to heat a portion of the
reverse transcription module to produce an inactivation temperature
zone within the second reaction volume; and produce a flow of the
input solution within the second reaction volume such that a volume
of the input solution is heated within the inactivation temperature
zone to inactivate an enzyme within the input solution.
66. The method of claim 65, wherein the volume of the input
solution is at least 10 microliters.
67. The method of claim 66, wherein the volume of the input
solution is produced within five minutes or less.
68. The method of claim 65, wherein the second reaction volume is a
serpentine flow path.
69. The method of claim 65, wherein a wall of the reverse
transcription module that defines the second reaction volume has a
surface area, a ratio of the surface area to the second reaction
volume being greater than about 10 cm-1.
70. The method of claim 65, wherein the volume of the input
solution is heated to an inactivation temperature of between about
57 degrees Celsius and about 100 degrees Celsius for a time period
of at least about 15 seconds.
71.-73. (canceled)
74. The method of claim 65, wherein the portion of the reverse
transcription module is a second portion, the actuating the
molecular diagnostic test device further causes the molecular
diagnostic test device to: heat a first portion of the reverse
transcription module to produce a lysing temperature zone within
the second reaction volume, the flow of the input solution within
the second reaction volume being such that the volume of the input
solution is heated within the lysing temperature zone to lyse a
biological entity within the volume of the input solution.
75.-78. (canceled)
79. The method of claim 65, wherein the actuating the molecular
diagnostic test device further causes the molecular diagnostic test
device to: heat a portion of an amplification module within the
molecular diagnostic test device to amplify a nucleic acid from the
plurality of cDNA molecules to produce an output containing a
target amplicon; and convey the output to a detection module of the
molecular diagnostic test device.
80. The method of claim 79, further comprising: reading from the
molecular diagnostic test device a signal indicating a presence of
the target amplicon; and discarding, after the reading, the
molecular diagnostic test device.
81. An apparatus, comprising: a housing; a sample input module
defining an input reservoir configured to receive a biological
sample, the biological sample containing a biological entity; a
reverse transcription module disposed within the housing, the
reverse transcription module including a heater and first flow
member, the first flow member defining a first volume and a second
volume, the first volume configured to receive an input solution
containing at least the biological sample and a lysis buffer, the
first volume further containing a lyophilized reverse transcription
reagent, the heater coupled to the first flow member and configured
to convey thermal energy into the second volume to A) reverse
transcribe at least a portion a plurality of ribonucleic acid (RNA)
molecules released from the biological sample to produce to convert
the plurality of RNA molecules into a plurality of complementary
DNA (cDNA) molecules and B) inactivate an enzyme within the input
solution or within the lyophilized reverse transcription reagent
when a volume of the input solution flows through the second
volume; and an amplification module disposed within the housing,
the amplification module including a second flow member configured
to receive the volume of the input solution from the reverse
transcription module, the amplification module configured to
amplify a nucleic acid molecule from the plurality of cDNA
molecules within the volume of the input solution to produce an
output containing a target amplicon.
82. The apparatus of claim 81, wherein the second volume of the
reverse transcription module is a serpentine flow path.
83. The apparatus of claim 81, wherein a wall of the reverse
transcription module that defines the second volume has a surface
area, a ratio of the surface area to the second volume being
greater than about 10 cm-1.
84. The apparatus of claim 81, wherein: the first volume is in
fluid communication with the second volume; and the reverse
transcription module defines a vent opening into the first
volume.
85. (canceled)
86. The apparatus of claim 81, wherein: the heater is a first
heater; the second flow member defines an amplification flow path;
and the amplification module includes a second heater different
from the first heater, the second heater coupled to the second flow
member and configured to convey thermal energy into the
amplification flow path to amplify the plurality of cDNA
molecules.
87. The apparatus of claim 81, further comprising: a fluid pump
disposed within the housing, the fluid pump configured to produce a
flow of the input solution from the reverse transcription module to
the amplification module.
88.-94. (canceled)
95. The apparatus of claim 22, wherein the apparatus is a
self-contained device configured to operate without any external
instrument.
96. The apparatus of claim 24, further comprising: a controller
disposed within the housing, the controller implemented in at least
one of a memory or a processor, the controller including a thermal
control module configured to produce a thermal control signal to
adjust an output of the heater, a power source being electrically
isolated from the processor when the reagent actuator is in the
first position, the power source being electrically coupled to at
least one of the processor or the amplification module when the
reagent actuator is in the second position.
97. The apparatus of claim 22, further comprising: a fluid pump
disposed within the housing, the fluid pump configured to generate,
within the housing, a force that causes a flow of the output
produced by the amplification module.
98. The apparatus of claim 81, wherein the apparatus is a
self-contained device configured to operate without any external
instrument.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/357,306, filed Jun. 30, 2016, which application
is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The embodiments described herein relate to methods and
devices for molecular diagnostic testing. More particularly, the
embodiments described herein relate to disposable, self-contained
devices and methods for molecular diagnostic testing. Particular
embodiments described herein relate to disposable, self-contained
devices and methods for purifying, reverse transcribing and
detecting nucleic acids.
[0003] There are over one billion infections in the U.S. each year,
many of which are treated incorrectly due to inaccurate or delayed
diagnostic results. Many known point of care (POC) tests have poor
sensitivity (30-70%), while the more highly sensitive tests, such
as those involving the specific detection of nucleic acids or
molecular testing associated with a pathogenic target, are only
available in laboratories. Thus, approximately ninety percent of
the current molecular diagnostics testing is practiced in
centralized laboratories. Known devices and methods for conducting
laboratory-based molecular diagnostics testing, however, require
trained personnel, regulated infrastructure, and expensive, high
throughput instrumentation. Known laboratory instrumentation is
often purchased as a capital investment along with a regular supply
of consumable tests or cartridges. Known high throughput laboratory
equi.mu.ment generally processes many (96 to 384 and more) samples
at a time, therefore central lab testing is done in batches. Known
methods for processing typically include processing all samples
collected during a time period (e.g., a day) in one large run, with
a turn-around time of hours to days after the sample is collected.
Moreover, such known instrumentation and methods are designed to
perform certain operations under the guidance of a skilled
technician who adds reagents, oversees processing, and moves sample
from step to step. Thus, although known laboratory tests and
methods are very accurate, they often take considerable time, and
are very expensive.
[0004] There are limited testing options available for testing done
at the point of care ("POC"), or in other locations outside of a
laboratory. Known POC testing options tend to be single analyte
tests with low analytical quality. These tests are used alongside
clinical algorithms to assist in diagnosis, but are frequently
verified by higher quality, laboratory tests for the definitive
diagnosis. Thus, neither consumers nor physicians are enabled to
achieve a rapid, accurate test result in the time frame required to
"test and treat" in one visit. As a result doctors and patients
often determine a course of treatment before they know the
diagnosis. This has tremendous ramifications: antibiotics are
either not prescribed when needed, leading to infections; or
antibiotics are prescribed when not needed, leading to new
antibiotic-resistant strains in the community. Moreover, known
systems and methods often result in diagnosis of severe viral
infections, such as H1N1 swine flu, too late, limiting containment
efforts. In addition, patients lose much time in unnecessary,
repeated doctor visits.
[0005] Thus, a need exists for improved devices and methods for
molecular diagnostic testing. In particular, a need exists for an
affordable, easy-to-use test that will allow healthcare providers
and patients at home to diagnose infections accurately and quickly
so they can make better healthcare decisions.
SUMMARY OF THE INVENTION
[0006] In one aspect, a molecular diagnostic test device includes a
housing, a reverse transcription module, an amplification module
and a detection module. The reverse transcriptase module is
configured to receive an input sample and includes a heater such
that the reverse transcription module can perform a reverse
transcriptase polymerase chain reaction (RT-PCR) on the input
sample. The amplification module is configured to receive a cDNA
sample from the reverse transcription module. The amplification
module includes a heater such that the amplification module can
perform a polymerase chain reaction (PCR) on the input sample. The
detection module is configured to receive an output from the
amplification module and a reagent formulated to produce a signal
that indicates a presence of a target amplicon within the input
sample. The reverse transcription module, amplification module and
the detection module are integrated within the housing such that
the molecular diagnostic test device is a handheld device.
[0007] In some cases, the signal is a non-fluorescent signal. In
some cases, the signal is a visible signal characterized by a color
associated with the presence of the target amplicon; and the
detection module includes a detection surface from which the
visible signal is produced, the detection surface visible via a
detection opening defined by the housing. In some cases, the signal
is a visible signal characterized by a color associated with the
presence of the target amplicon, the reagent formulated such that
the visible signal remains present for at least about 30
minutes.
[0008] In some cases, the molecular diagnostic test device further
comprises a power source disposed within the housing and configured
to supply power to the amplification module, the power source
including a DC battery having a nominal voltage of about 9V, the
power source having a capacity of less than about 1200 mAh.
[0009] In some cases, the molecular diagnostic test device further
comprises a power source disposed within the housing; and a reagent
module disposed within the housing, the reagent module including a
sealed volume within which the reagent is contained, the reagent
module including a reagent actuator configured to convey the
reagent into a holding chamber fluidically coupled to the detection
module when the reagent actuator is moved from a first position to
a second position, the power source being electrically isolated
from the amplification module when the reagent actuator is in the
first position, the power source being electrically coupled to at
least one of a processor or the amplification module when the
reagent actuator is in the second position. In some cases, the
molecular diagnostic test device further comprises a sample input
module disposed within the housing, the sample input module
including an inlet port, an outlet port, the inlet port configured
to receive the input sample; and a sample actuator configured to
convey the input sample via the outlet port and through a filter
assembly when the sample actuator is moved from a first position to
a second position, the sample actuator configured to remain locked
in the second position. In some cases, the sample actuator is in a
fixed position relative to at least one of the amplification module
or the detection module when the sample actuator is in the second
position. In some cases, the sample actuator is a non-electronic
actuator configured to move irreversibly from the first position to
the second position. In some cases, the molecular diagnostic test
device is configured for one and only one use and is
disposable.
[0010] In another aspect an apparatus comprises a housing defining
a detection opening; a reverse transcription module disposed within
the housing, the reverse transcription module including a flow
member and a heater, the flow member defining an reverse
transcription flow path having an inlet portion configured to
receive a sample, the heater fixedly coupled to the flow member
such that the heater and the amplification flow path intersect at
multiple locations; an amplification module disposed within the
housing, the amplification module including a flow member and a
heater, the flow member defining an amplification flow path having
an inlet portion configured to receive a sample, the heater fixedly
coupled to the flow member such that the heater and the
amplification flow path intersect at multiple locations; a reagent
module disposed within the housing, the reagent module containing a
substrate formulated to catalyze the production of a signal by a
signal molecule associated with a target amplicon; and a detection
module defining a detection channel in fluid communication with an
outlet portion of the amplification flow path and the reagent
module, the detection module including a detection surface within
the detection channel, the detection surface configured to retain
the target amplicon, the detection module disposed within the
housing such that the detection surface is visible via the
detection opening of the housing. In some cases, the amplification
and/or reverse transcription flow path is a serpentine flow path,
and the heater is a linear heater irreversibly coupled to the flow
member. In some cases, the amplification and/or reverse
transcription flow path is a serpentine flow path; the heater is a
heater assembly including a first linear heater coupled to a first
end portion of the flow member, a second linear heater coupled to a
second end portion of the flow member, a third linear heater
coupled to a central portion of the flow member, the heater
assembly coupled to of a first side the flow member via an adhesive
bond.
[0011] In some cases, the apparatus further comprises a power
source disposed within the housing and configured to supply power
to the heater, the power source having a nominal voltage of about 9
VDC and a capacity of less than about 1200 mAh. In some cases, the
apparatus further comprises a power module removably coupled to the
housing, the power module including a power source having a nominal
voltage of about 9 VDC and a capacity of less than about 1200 mAh,
the power module including an electronic circuit electrically
coupled to the heater when the power module is coupled to the
housing.
[0012] In some cases, the apparatus further comprises a power
source having a nominal voltage of about 9 VDC and a capacity of
less than about 1200 mAh; and an isolation member removably coupled
to the housing, the power source being electrically isolated from
the heater when the isolation member is coupled to the housing, the
power source being electrically coupled to the heater when the
isolation member is removed from the housing,
[0013] the reagent module including a reagent actuator configured
to release the substrate into a holding chamber when the reagent
actuator is moved from a first position to a second position, the
movement of the isolation member being limited when the reagent
actuator is in the first position.
[0014] In some cases, the apparatus further comprises a power
source disposed within the housing, the reagent module including a
reagent actuator configured to release the substrate into a holding
chamber when the reagent actuator is moved from a first position to
a second position, the power source being electrically isolated
from the heater when the reagent actuator is in the first position,
the power source being electrically coupled to the heater when the
reagent actuator is in the second position. In some cases, the
apparatus further comprises a controller disposed within the
housing, the controller implemented in at least one of a memory or
a processor, the controller including a thermal control module
configured to produce a thermal control signal to adjust an output
of the heater.
[0015] In some cases, the signal is a visible signal characterized
by a color associated with the presence of the target amplicon; and
the detection channel has a width of at least about 4 mm. In some
cases, the housing includes a mask portion configured to surround
at least a portion of the detection opening, the mask portion
configured to enhance visibility of the detection surface through
the detection opening.
[0016] In some cases, the reagent module includes a reagent
formulated to produce the signal; and the signal is a
non-fluorescent visible signal characterized by a color associated
with the presence of the target amplicon, the reagent formulated
such that the visible signal remains present for at least about 30
minutes.
[0017] In another aspect an apparatus comprises a housing; a sample
preparation module disposed within the housing and configured to
receive an input sample, the sample preparation module including a
filter assembly; a reverse transcription module disposed within the
housing and configured to receive an output from the sample
preparation module, the reverse transcription module including a
flow member and a heater, the flow member defining an reverse
transcription flow path having an inlet portion configured to
receive a sample, the heater fixedly coupled to the flow member
such that the heater and the amplification flow path intersect at
multiple locations; an amplification module disposed within the
housing and configured to receive an output from the reverse
transcription module, the amplification module including a flow
member and a heater, the flow member defining a serpentine flow
path, the heater coupled to the flow member, the amplification
module configured perform a polymerase chain reaction (PCR) on the
output from the sample preparation module; and a detection module
disposed within the housing and configured to receive an output
from the amplification module, wherein the apparatus is configured
for one-time use. In some cases, the detection module is configured
to receive a reagent formulated to produce a colorimetric signal
that indicates a presence of a target organism in the input sample.
In some cases, the apparatus further comprises a sample actuator
configured to produce a force to convey the input sample through
the filter assembly when the sample actuator is moved from a first
position to a second position, the sample actuator configured to
remain locked in the second position, the sample actuator including
a locking shoulder configured to matingly engage a portion of the
housing to maintain the sample actuator in the second position. In
some cases, the sample preparation module is fixedly coupled within
the housing. In some cases, the detection module is fixedly coupled
within the housing and includes a detection surface from which a
colorimetric signal that indicates a presence of a target organism
in the input sample is produced, the detection surface visible via
a detection opening defined by the housing.
[0018] In some cases, the apparatus further comprises a fluid
transfer module disposed within the housing, the fluid transfer
module defining an internal volume within which the output of the
sample preparation module flows when the fluid transfer module is
actuated, the fluid transfer module configured to convey the output
of the sample preparation module from the internal volume to the
amplification module, the fluid transfer module being fixedly and
fluidically coupled to the sample preparation module. In some
cases, the fluid transfer module includes a plunger movably
disposed within the internal volume such that movement of the
plunger conveys the output of the sample preparation module from
the internal volume to the amplification module. In some cases, the
apparatus further comprises a power source disposed within the
housing and configured to supply power to the amplification module,
the power source having a capacity of less than about 1200 mAh. In
some cases, the sample preparation module includes a wash container
containing a gas wash and a liquid wash, the sample preparation
assembly configured to convey the gas wash and the liquid wash
through the filter assembly in series, further comprising: a wash
actuator configured to produce a force to convey the gas wash
through the filter assembly at a first time and the liquid wash
through the filter assembly at a second time after the first time
when the wash actuator is moved from a first position to a second
position.
[0019] In some cases, the heating element can heat a liquid in the
mixing chamber to a temperature between 20 C and 100 C. In some
cases, the heating element can heat a liquid in the mixing chamber
to a temperature between 20 C and 50 C. In some cases, the heating
element can heat a liquid in the mixing chamber to a temperature
between 85 C and 95 C. In some cases, the heating element can hold
a liquid in the mixing chamber at a constant temperature between 20
C and 50 C. In some cases, the heating element can hold a liquid in
the mixing chamber at a constant temperature between 85 C and 95 C.
In some cases, the heating element can hold a liquid in the mixing
chamber at a constant temperature for a time between 0.1 to 24
hours. In some cases, the heating element can hold a liquid in the
mixing chamber at a constant temperature for a time between 0.1 to
1 hour. In some cases, the heating element can hold a liquid in the
mixing chamber at a constant temperature for a time between 1
second and 30 minutes. In some cases, the heating element can hold
a liquid in the mixing chamber at a constant temperature for a time
between 1 second and 10 minutes. In some cases, the reverse
transcription chamber of step (b) further comprises a mixing
chamber and a serpentine channel. In some cases, the mixing chamber
can hold a volume between 10 ul and 10 mls In some cases, the
mixing chamber can hold a volume between 10 ul and 1 ml. In some
cases, the mixing chamber can hold a volume of 300 ul. In some
cases, the serpentine channel is designed to have a cross-section
with an aspect ratio (channel height to width) to maximize the area
in contact with heater allowing efficient heat coupling to the
fluid. In some cases, the device is designed to perform and analyze
multiplexed PCRs. In some cases, the reverse transcription module
further comprises a lyophilized pellet comprising reverse
transcriptase enzyme and reagents. In some cases, the reverse
transcription module contains a reagent chamber containing reverse
transcriptase enzyme and reagents required for a reverse
transcriptase polymerase chain reaction. In some cases, the reverse
transcriptase enzyme and reagents are present as a lyophilized
pellet. In some cases, the reverse transcriptase enzyme and
reagents are present with the DNA polymerase enzyme and PCR
reagents.
[0020] In another aspect, a method for DNA preparation comprises
obtaining a biological sample comprising one or more biological
entities comprising RNA; capturing said one or more biological
entities on a filter; eluting said one or more biological entities
from said filter; and lysing said one or more biological entities,
incubating the lysed biological entities with a reverse
transcriptase enzyme and sufficient reagents to perform a reverse
transcription reaction, thereby preparing a plurality of DNA
molecules therefrom, wherein said method prepares said DNA
molecules from said one or more biological entities within 10
minutes or less at a quality sufficient to successfully perform a
polymerase chain reaction (PCR).
[0021] In some cases, the method further comprises that the filter
consists of two filter membranes, a first filter membrane and a
second filter membrane with a smaller pore size than the first
filter membrane.
[0022] In some cases, the method further comprises a wash step,
whereby once the biological entities are captured on the filter the
filter and biological entities are washed with an air wash.
[0023] In another aspect, a method for DNA preparation comprises
obtaining a biological sample comprising one or more biological
entities, wherein the biological entities comprise RNA; lysing said
one or more biological entities, thereby releasing a plurality of
RNA molecules therefrom; and performing a reverse transcriptase
reaction on the released RNA molecules to produce a plurality of
DNA molecules, wherein said method extracts said nucleic acid
molecules from said one or more biological entities within 5
minutes or less at a quality sufficient to successfully perform a
polymerase chain reaction (PCR). In some cases, the method is
performed by a handheld device. In some cases, a quality sufficient
to successfully perform a polymerase chain reaction comprises
nucleic acid molecules which amplify with at least 70% efficiency
as determined by a qPCR standard curve. In some cases, the method
produces at least 100 .mu.L of a solution containing the nucleic
acid molecules. In some cases, the method produces at least 300 of
a solution containing the nucleic acid molecules. In some cases,
the method produces at least 500 of a solution containing the
nucleic acid molecules. In some cases, the method further comprises
catching biological entities on a filter and subjecting the
biological entities and filter to an air wash. In some cases, the
biological entities are washed with a volume of air sufficient to
dry the filter. In some cases, the biological entities are washed
with at least about 1.5 mL of air.
[0024] In another aspect, a device is configured to perform a
method as described herein, wherein said device comprises an input
port, configured to receive said biological sample comprising one
or more biological entities; a holding tank, operably coupled to
said input port, an inactivation section, and containing a heating
element; and an output port. In some cases, the device further
comprises a permanent vent. In some cases, the holding tank further
comprises an electrical probe which can sense the presence of
liquid in the holding tank. In some cases, the inactivation chamber
comprises a serpentine path.
[0025] In another aspect, a method of DNA preparation comprises
conveying a biological sample comprising RNA into a sample input
module of a molecular diagnostic test device; and actuating the
molecular diagnostic test device to: lyse the biological sample in
a lysing module, convey the biological sample from the lysing
module to a reverse transcription module, the reverse transcription
module including a heater and defining a first reaction volume and
a second reaction volume, and further comprising lyophilized
reagents for a reverse transcription reaction; maintain an input
solution containing the biological sample and the reagents for
reverse transcription within the first reaction module to reverse
transcribe at least a portion of the biological sample thereby
producing a plurality of DNA molecules; activate the heater to heat
a portion of the lysing module to produce an inactivation
temperature zone within the second reaction volume; and produce a
flow of the input solution within the second reaction volume such
that a volume of the input solution is heated within the
inactivation temperature zone to inactivate an enzyme within the
input solution. In some cases, the volume of the input solution is
at least 10 microliters. In some cases, the volume of the input
solution is produced within five minutes or less. In some cases,
the second reaction volume is a serpentine flow path. In some
cases, a wall of the lysing module that defines the second reaction
volume has a surface area, a ratio of the surface area to the
second reaction volume being greater than about 10 cm-1. In some
cases, the volume of the input solution is heated to an
inactivation temperature of between about 57 degrees Celsius and
about 100 degrees Celsius for a time period from about 15 seconds.
In some cases, the flow of the input solution is such that the
volume of the input solution is heated to an inactivation
temperature of between about 92 degrees Celsius and about 98
degrees Celsius for a time period of at least about 25 seconds. In
some cases, the first reaction volume is in fluid communication
with the second reaction volume; and the reverse transcription
module defines a vent opening into the first reaction volume. In
some cases, the volume of the input solution is heated to an
inactivation temperature of at least about 95 degrees Celsius; and
the input solution within the first reaction module contains at
least one of a salt or a sugar formulated to raise a boiling
temperature of the input solution. In some cases, the portion of
the reverse transcription module is a second portion, the actuating
the molecular diagnostic test device further causes the molecular
diagnostic test device to: heat a first portion of the lysing
module to produce a lysing temperature zone within the second
reaction volume, the flow of the input solution within the second
reaction volume being such that the volume of the input solution is
heated within the lysing temperature zone to lyse a biological
entity within the volume of the input solution. In some cases, the
actuating the molecular diagnostic test device causes the molecular
diagnostic test device to: convey the biological sample from the
sample input module through a filter to retain a biological entity
with the biological sample on the filter; and produce a flow of an
elution buffer through the filter to produce the input solution and
convey the input solution to the lysing module. In some cases, the
actuating the molecular diagnostic test device includes moving a
sample actuator to produce a pressure within the sample input
module to convey the biological sample from the sample input module
towards the lysing module. In some cases, the sample actuator is a
non-electronic actuator. In some cases, the actuating the molecular
diagnostic test device further causes the molecular diagnostic test
device to: receive an electronic signal from a sensor within the
reverse transcription module, the electronic signal indicating the
presence of the input solution within the first reaction module;
and activate the heater in response to the electronic signal. In
some cases, the actuating the molecular diagnostic test device
further causes the molecular diagnostic test device to: heat a
portion of an amplification module within the molecular diagnostic
test device to amplify a nucleic acid from the plurality of nucleic
acid molecules to produce an output containing a target amplicon;
and convey the output to a detection module of the molecular
diagnostic test device. In some cases, the method further comprises
viewing a visible signal indicating a presence of the target
amplicon; and discarding, after the viewing, the molecular
diagnostic test device.
[0026] In another aspect an apparatus, comprises a housing; a
sample input module defining an input reservoir configured to
receive a biological sample, the biological sample containing a
biological entity; a lysing module disposed within the housing, the
lysing module including a heater and first flow member, the first
flow member defining a first volume and a second volume, the first
volume configured to receive an input solution containing at least
the biological sample and a lysis buffer, the heater coupled to the
first flow member and configured to convey thermal energy into the
second volume to A) lyse at least a portion of the biological
sample thereby releasing a plurality of nucleic acid molecules and
B) inactivate an enzyme within the input solution when a volume of
the input solution flows through the second volume; a reverse
transcription module disposed within the housing, the reverse
transcription module including a heater and first flow member, the
first flow member defining a first volume and a second volume, the
first volume configured to receive an input solution containing at
least the biological sample and a lysis buffer, the first volume
further containing lyophilized reagents for a reverse transcription
reaction, the heater coupled to the first flow member and
configured to convey thermal energy into the second volume to A)
reverse transcribe at least a portion of the biological sample
thereby releasing a plurality of nucleic acid molecules and B)
inactivate an enzyme within the input solution or within the
lyophilized reverse transcription reagents when a volume of the
input solution flows through the second volume; and an
amplification module disposed within the housing, the amplification
module including a second flow member configured to receive the
volume of the input solution from the lysing module, the
amplification module configured to amplify a nucleic acid molecule
from the plurality of nucleic acid molecules within the volume of
the input solution to produce an output containing a target
amplicon. In some cases, the second volume of the reverse
transcription module is a serpentine flow path. In some cases, a
wall of the reverse transcription module that defines the second
volume has a surface area, a ratio of the surface area to the
second reaction volume being greater than about 10 cm-1. In some
cases, the first volume is in fluid communication with the second
reaction volume; and the reverse transcription module defines a
vent opening into the first volume. In some cases, the lysing
module includes a sensor disposed within the first volume, the
sensor configured to produce an electronic signal indicating the
presence of the input solution within the first module, the heater
activated in response to the electronic signal. In some cases, the
heater is a first heater; the second flow member defines an
amplification flow path; and the amplification module includes a
second heater different from the first heater, the second heater
coupled to the second flow member and configured to convey thermal
energy into the amplification flow path to amplify the nucleic acid
molecule from the plurality of nucleic acid molecules. In some
cases, the apparatus further comprises a non-electronic sample
actuator to produce a pressure within the sample input module to
convey the biological sample from the sample input module towards
the lysing module; and a fluid pump disposed within the housing,
the fluid pump configured to produce a flow of the input solution
from the lysing module to the amplification module. In some cases,
the flow of the input solution from the lysing module to the
amplification module is in a first direction; and the lysing module
includes a check valve to configured to prevent a flow of the input
solution in a second direction. A device comprising a holding tank
which contains two electrical probes which may be used to determine
the electrical resistance of the fluid within the holding tank,
thus determining whether liquid has entered the holding tank.
[0027] In another aspect, an apparatus comprises a reverse
transcription module disposed within a molecular diagnostic test
device, the reverse transcription module including a heater and a
flow member, the flow member defining a first volume and a second
volume, the first volume containing a lyophilized reverse
transcriptase enzyme and configured to receive an input solution
containing at least a biological sample, the heater coupled to the
flow member and configured to convey thermal energy into the
reverse transcription module to facilitate a thermal reaction on
the input solution when a volume of the input solution flows
through the second volume; and a sensor at least partially disposed
within the first volume the sensor configured to produce a signal
when the input solution is within the first volume, a portion of
the molecular diagnostic test device being actuated in response to
the signal. In some cases, the sensor includes a first electrode
and a second electrode, the first electrode disposed within the
first volume, the second electrode disposed within the second
volume, spaced apart from the first electrode, the sensor
configured to determine an electrical resistance of the input
solution between the first electrode and the second electrode and
produce the signal associated with the electrical resistance. In
some cases, the heater is actuated in response to the signal. In
some cases, the apparatus further comprises an amplification module
disposed within the housing, the amplification module including an
amplification flow member configured to receive the volume of the
input solution from the reverse transcription module, the
amplification module configured to amplify a nucleic acid molecule
from a plurality of nucleic acid molecules within the volume of the
input solution to produce an output containing a target amplicon,
the amplification module being actuated in response to the signal.
In another aspect, a method for increasing the concentration of a
biological entity in a liquid comprises obtaining a plurality of
hydrogel particles functionalized with affinity baits for said
biological entity; incubating a first volume of the liquid
containing the biological entity with the hydrogel particles;
flowing the liquid containing the biological entity and the
hydrogel particles through a filter with a pore size such that the
hydrogel particles cannot pass through the filter; and eluting the
hydrogel particles and bound biological entity from the filter in a
second volume of liquid, wherein the second volume of liquid is
smaller than the first volume of liquid, thus increasing the
concentration of the biological entity in the liquid.
INCORPORATION BY REFERENCE
[0028] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0030] FIG. 1 depicts data generated from a real-time PCR reaction
performed on DNA extracted from clinical samples utilizing the
methods provided herein.
[0031] FIG. 2 depicts data generated from a real-time PCR reaction
performed on DNA extracted from clinical samples utilizing standard
DNA extraction methods.
[0032] FIG. 3 depicts a comparison of data generated from a
real-time PCR reaction performed on DNA extracted from a clinical
sample positive for both N. gonorrhoeae and C. trachomatis (Sample
122) and a clinical sample positive for N. gonorrhoeae (Sample 117)
utilizing the methods provided herein versus standard DNA
extraction methods.
[0033] FIG. 4 depicts a comparison of data generated from a
real-time PCR reaction performed on DNA extracted from a clinical
sample positive for both N. gonorrhoeae and C. trachomatis (Sample
122) and a clinical sample positive for N. gonorrhoeae (Sample 117)
utilizing the methods provided herein versus standard DNA
extraction methods.
[0034] FIG. 5 depicts a comparison of data generated from a
real-time PCR reaction performed on DNA extracted from a clinical
sample positive for both N. gonorrhoeae and C. trachomatis (Sample
122), a clinical samples positive for C. trachomatis (Samples 101
and 108) utilizing the methods provided herein versus standard DNA
extraction methods.
[0035] FIG. 6 depicts a comparison of data generated from a
real-time PCR reaction performed on DNA extracted from a clinical
sample positive for both N. gonorrhoeae and C. trachomatis (Sample
122) and clinical samples positive for C. trachomatis (Samples 101
and 108) utilizing the methods provided herein versus standard DNA
extraction methods.
[0036] FIG. 7 depicts a comparison of data generated from a
real-time PCR reaction performed on N. gonorrhoeae DNA utilizing
different sets of primers.
[0037] FIG. 8 depicts a comparison of data generated from a
real-time PCR reaction performed on C. trachomatis DNA utilizing
different sets of primers.
[0038] FIG. 9 depicts data generated from a real-time PCR reaction
performed on N. gonorrhoeae DNA spiked into a sample and PCR
mixture to test for sample inhibition.
[0039] FIG. 10 is a schematic illustration of a molecular
diagnostic test device, according to an embodiment, which can
perform the methods described herein.
[0040] FIG. 11 is an exploded view of a molecular diagnostic test
device, according to an embodiment, which can perform the methods
described herein.
[0041] FIG. 12 depicts an example of a sample preparation device
amenable to performing the methods as described herein.
[0042] FIG. 13 is a perspective view of a lysing module according
to an embodiment, which is amenable to performing the methods as
described herein.
[0043] FIG. 14 is an exploded view of the lysing module shown in
FIG. 13.
[0044] FIG. 15 is a top view of a portion of the lysing module
shown in FIG. 13.
[0045] FIG. 16 is a cross-sectional view of the lysing module shown
in FIG. 13.
[0046] FIGS. 17 and 18 is are perspective views of a lysing module
according to an embodiment, which can perform any of the methods
described herein.
[0047] FIG. 19 is a bottom view of the lysing module shown in FIGS.
17 and 18.
[0048] FIG. 20 is a cross-sectional view of the lysing module shown
in FIGS. 17 and 18 taken along line X.sub.1-X.sub.1 in FIG. 19.
[0049] FIG. 21 is a cross-sectional view of the lysing module shown
in FIGS. 17 and 18 taken along line X.sub.2-X.sub.2 in FIG. 19.
[0050] FIG. 22 is a perspective view of a portion of the lysing
module shown in FIGS. 17 and 18.
[0051] FIG. 23 is a schematic illustration of a portion of a
molecular diagnostic test device, according to an embodiment, which
can perform the methods described herein.
[0052] FIG. 24 is a schematic illustration of a molecular
diagnostic test device, according to an embodiment, which can
perform the methods described herein.
[0053] FIG. 25 illustrates the results of a PCR reaction performed
upon DNA extracted using the methods of this disclosure.
[0054] FIG. 26 illustrates the results of a PCR reaction performed
upon DNA extracted using the methods of this disclosure.
[0055] FIG. 27 illustrates the results of a PCR reaction performed
upon DNA extracted using the methods of this disclosure.
[0056] FIG. 28 illustrates a block diagram of a device including a
reverse transcription module.
[0057] FIG. 29 illustrates a temperature profile in a reverse
transcription module.
[0058] FIG. 30 illustrates a possible chamber design for a reverse
transcription module.
[0059] FIG. 31 illustrates the bottom view of a possible chamber
design for a reverse transcription module.
[0060] FIG. 32 illustrates an example of a functionalized
nanoparticle.
[0061] FIG. 33 illustrates a proposed model of functionalized
nanoparticle binding to viruses.
[0062] FIG. 34 illustrates a block diagram of a device including a
reverse transcription module.
[0063] FIG. 35 is a schematic illustration of a portion of a
molecular diagnostic test device, according to an embodiment, which
can perform the methods described herein.
[0064] FIG. 36 illustrates capture of viral nucleic acid with
affinity particles
[0065] FIG. 37 illustrates capture of infectious viral particles
with affinity particles.
DETAILED DESCRIPTION OF THE INVENTION
[0066] Disclosed herein are devices and methods for the preparation
of nucleic acid molecules for downstream applications. In some
cases, the devices and methods are utilized for the extraction of
nucleic acid molecules from a biological sample. In some cases, the
devices and methods are utilized for the purification of nucleic
acid molecules from a biological sample. In some cases, the devices
and methods are utilized to produce and detect a cDNA from an RNA
isolated from a biological sample. The devices described herein may
include self-contained, handheld devices. The devices described
herein may include one or more components that aid in the
extraction, purification, and/or processing of a biological sample
and the nucleic acids contained therein. In some cases, the methods
include the use of a device that includes one or more components
that aid in the extraction, purification, and/or processing of a
biological sample and the nucleic acids contained therein. In some
cases, the processing of a biological sample may include a reverse
transcription step which may be achieved by a reverse
transcriptase.
[0067] In one aspect, a method is provided for nucleic acid
extraction. The method may include one or more steps including: (a)
obtaining a biological sample comprising one or more biological
entities; (b) capturing the one or more biological entities on a
filter; (b) washing the filter with a wash solution and/or air; (c)
eluting the one or more biological entities from the filter; and
(d) lysing the one or more biological entities, thereby releasing a
plurality of nucleic acid molecules therefrom. In some cases, the
wash solution comprises bovine serum albumin and/or a detergent. In
some cases, the wash solution comprises about 0.1% to 5% bovine
serum albumin. In some cases, the wash solution comprises about
0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, or 5%
bovine serum albumin. In some cases, the wash solution comprises
about 0.1% to 20% detergent. In some cases, the wash solution
comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%
detergent. In some cases, the detergent is Tween-20. In some
embodiments the method may not require use of a filter. In other
embodiments the method may use a filter but not require a wash
solution. In some cases, the method further includes a step of
reverse transcribing an RNA molecule to produce a cDNA molecule. In
some further cases, the method includes a preliminary step for
increasing the concentration of one or more biological entities in
the sample. This step may involve the use of affinity beads
designed to bind to pathogens or analytes in the sample. The
affinity beads may be nanoparticles or microparticles,
(functionalized nanoparticles or functionalized
microparticles).
[0068] In some cases, the method involves obtaining or providing a
biological sample. The biological sample can be derived from a
non-cellular entity comprising polynucleotides (e.g., a virus) or
from a cell-based organism (e.g., member of archaea, bacteria, or
eukarya domains).
[0069] Generally, the biological sample will contain one or more
biological entities that comprise one or more polynucleotides or
nucleic acid molecules. A "nucleic acid molecule", "nucleic acid"
or "polynucleotide" may be used interchangeably throughout and may
refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
including known analogs or a combination thereof unless otherwise
indicated. Nucleic acid molecules to be profiled herein can be
obtained from any source of nucleic acid. The nucleic acid molecule
can be single-stranded or double-stranded. In some cases, the
nucleic acid molecules are RNA. RNA can include, but is not limited
to, mRNAs, tRNAs, snRNAs, rRNAs, retroviruses, small non-coding
RNAs, microRNAs, polysomal RNAs, pre-mRNAs, intronic RNA, viral
RNA, cell free RNA and fragments thereof. The non-coding RNA, or
ncRNA can include snoRNAs, microRNAs, siRNAs, piRNAs and long nc
RNAs. In some cases, the nucleic acid molecules are DNA. The DNA
can be mitochondrial DNA, complementary DNA (cDNA), or genomic DNA.
In some cases, the nucleic acid molecules are genomic DNA (gDNA).
The DNA can be plasmid DNA, cosmid DNA, bacterial artificial
chromosome (BAC), or yeast artificial chromosome (YAC). The DNA can
be derived from one or more chromosomes. For example, if the DNA is
from a human, the DNA can derived from one or more of chromosomes
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, X, or Y. The source of nucleic acid for use in the
methods and compositions described herein can be a sample
comprising the nucleic acid.
Concentrating One or More Biological Entities in the Sample
[0070] In some aspects, the methods involve capturing one or more
biological cells or biological entities (e.g., a virus) with a
capture particle or affinity bead. Various methodologies may be
used to capture and concentrate pathogens from biological fluids
(e.g., blood, plasma, homogenized tissue, urine). The capture
methods may be generic and bind to any cells or biological entities
in a sample, or may be specific to a class or type of biological
entity. In other cases, the capture methods may be specific to a
family of pathogens, for example a family of bacteria or viruses.
In some cases, the capture methods may be specific to a single
species of pathogen, for example a single species of bacteria or
virus. In some cases the capture methods may be designed to bind to
several related or unrelated pathogens. For example the capture
methods may be designed to bind one or more of the following
pathogens: Ebola virus, Sudan virus, Tai Forest virus, Bundibugyo
virus, Yersinia pestis, Zika virus, Plasmodium falciparum,
Leptospira interrogans, Dengue virus, Chikungunya virus,
Crimean-Congo hemorrhagic fever virus, and Lassa virus.
[0071] In some cases, the capturing and concentration of a
biological entity is achieved by use of a particle which the
biological entity adheres to. The particle may be made of any
substance. In some embodiments, the particle is a hydrogel
particle. In some examples the particle is a hydrogel particle
based on cross-linked N-isopropylacrylamide (NIPAm). The particle
may comprise a core with a porous coating. An example of such a
particle is shown in FIG. 33. In some cases, the particle may have
a porous coating which performs a size exclusion function limiting
the biological entities which may bind the particle.
[0072] The particle may be functionalized with a variety of
affinity baits to facilitate the binding and retention of
biological targets. In some cases, the functionalized particle may
be composed of a core containing high affinity aromatic baits,
surrounded by a sieving shell. Examples of aromatic baits include:
Cibacron Blue, Allylamine and Methacrylate. The outer shell may be
tailored for active exclusion of high abundance proteins. For
example, the outer shell may contain vinyl sulfonic acid for active
molecular sieving of high-abundance proteins. The functionalized
particles may be tailored to capture target analytes from a variety
of complex biological matrices, including blood, serum, plasma,
saliva and nasopharyngeal fluids. The target analytes may be
proteins, nucleic acids, viruses or bacteria. The functionalized
particles may capture live bacteria and intact viruses without
causing damage.
[0073] The functionalized particles may be nanoparticles. In some
cases the functionalized nanoparticles have an average diameter of
about 10-100, 20-40, 30-50 or 20-30 nm. In some embodiments, a
functionalized nanoparticle may have a diameter of about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, 50 or more than 50 nm In some embodiments, the
functionalized particles may be microparticles. In some cases the
functionalized microparticles have an average diameter of about
10-100, 20-40, 30-50 or 20-30 .mu.m.
[0074] In some instances, a functionalized microparticle may be
created by attaching one or more functionalized nanoparticles to a
larger particle. The larger particle may have a diameter of about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50 or more than 50 .mu.m. In some cases the
larger particle may have a diameter between about 1 and 10, 1 and
5, 5 and 10, 3 and 8, or 2 and 7 .mu.m. The larger particle may be
a hydrogel particle or a different type of particle. In some cases,
the larger particle is a polystyrene particle. A larger particle
may be bound to an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or more than 50
functionalized nanoparticles. The functionalized nanoparticles may
be covalently bound to the larger particle. In some cases, the
functionalized nanoparticle chemistry may incorporate amine
containing monomers into the hydrogel matrix.
[0075] To concentrate a biological entity within a sample one or
more functionalized nanoparticles or functionalized microparticles
designed to bind said biological entity may be added to the sample.
After incubation of the functionalized nanoparticles or
functionalized microparticles in the sample for a sufficient time
at a suitable temperature to allow binding of the biological
entity, the functionalized nanoparticles or functionalized
microparticles are extracted from the sample. In some cases, the
functionalized nanoparticles or functionalized microparticles are
extracted by flowing the sample through a filter with a pore size
smaller than the size of the particles. The functionalized
nanoparticles or functionalized microparticles and associated
biological entities may subsequently be washed off the filter and
nucleic acids may be released by lysis. In some embodiments
functionalized nanoparticles or functionalized microparticles are
added to a sample prior to processing the sample through a method
of device as described herein. In some embodiments, functionalized
microparticles will be lyophilized and put into sample collection
tubes, so upon collection of a sample into the tube, the
functionalized microparticles will hydrate and actively capture the
relevant biological entities. The sample and functionalized
microparticle mixture may be used directly in the methods and
devices described herein.
[0076] The incubation step for the functionalized microparticles
and the biological entities may be at least about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, 50, 55, or 60 minutes. In some cases the incubation step is
between 1 and 60, 1 and 30, 1 and 20, 1 and 15, 1 and 10 or 1 and 5
minutes. In some cases the incubation step is less than 1 minute.
In some cases, the incubation step is performed at room
temperature. In some cases, the incubation step is performed at a
temperature between about 15 and 80, 20 and 40, 20 and 30, 20 and
25, or 25 and 30.degree. C.
[0077] The functionalized nanoparticles or functionalized
microparticles may provide an enrichment of a biological entity in
a solution by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
than 30 fold. Using a method or device as described herein with a
functionalized microparticle may result in an increase in the
amount of nucleic acid extracted or prepared of about 2, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30 or more than 30 fold compared to the
same method or device without the functionalized microparticle.
Filter
[0078] In some aspects, the methods involve capturing one or more
biological cells or biological entities (e.g., a virus, or a
functionalized microparticle with trapped virus particles) present
in the biological sample on a filter membrane. The filter membrane
may be of any suitable material, non-limiting examples including
nylon, cellulose, polyethersulfone (PES), polyvinylidene difluoride
(PVDF), polycarbonate, borosilicate glass fiber and the like. In
some examples, the filter membrane is nylon. In some cases, the
filter membrane has an average pore size of about 0.2 .mu.m to
about 20 .mu.m. For example, the filter membrane may have an
average pore size of about 0.2 .mu.m, about 0.5 .mu.m, about 1
.mu.m, about 2 .mu.m, about 3 .mu.m, about 4 .mu.m, about 5 .mu.m,
about 6 .mu.m, about 7 .mu.m, about 8 .mu.m, about 9 .mu.m, about
10 .mu.m, about 11 .mu.m, about 12 .mu.m, about 13 .mu.m, about 14
.mu.m, about 15 .mu.m, about 16 .mu.m, about 17 .mu.m, about 18
.mu.m, about 19 .mu.m, about 20 .mu.m, or greater than 20 .mu.m. In
some examples, the surface of the filter membrane may be chemically
treated or coated in such a way as to improve the binding of a
biological cell or entity to the membrane. For example, without
limitation, the filter membrane may be treated with sodium
polyphosphate.
[0079] Clinical swab samples may contain mucus (or other
substances) which can lead to clogging of the filter used in sample
prep. If the filter is clogged then pressures may build up which
may lead to leaks in the fluidic path of the sample prep device
and/or tears or breaks in the capture filter itself. In some
examples a second filter may be provided which sits next to a first
filter. For example, a mesh screen may be placed on the input side
of the 5 micron nylon filter. This may reduce pressure from mucus
samples and also prevent the 5 micron nylon filter from breaking. A
mesh screen could also be placed on the exit side of the 5 micron
nylon filter which would also prevent the 5 micron nylon filter
from breaking, however this may not reduce the pressure required to
push a sample (mucus) through.
[0080] The mesh screen may be made from any plastic materials and
may contain pore sizes from 1 micron to 1000 microns. In some
embodiments the mesh screen may be a woven nylon mesh with 100
micron pores. The mesh screen is assembled into the housing that
also contains the 5 micron nylon filter. The second filter may have
a much larger pore size than the first filter and prevent clogging
of the first filter. For example the first filter may have a pore
size of about 0.1-20, 1-15, 1-10, 5-10, 1-5 or 0.1-1 .mu.m while
the second filter has a pore size of about 10-1000, 50-500,
100-500, 50-100, or 100-200 .mu.m. In one example the first filter
has a pore size of 5 .mu.m and the second filter has a pore size of
100 .mu.m. The mesh filter may also be made from non-woven
polypropylene. The mesh screen may have a thickness of about 150
.mu.m, 200 .mu.m or greater than 200 .mu.m. After the biological
cells or biological entities are captured on the filter membrane,
the filter membrane may be optionally washed with one or more wash
steps. The wash step may be utilized to, for example, remove any
undesired material from the membrane. In some cases, the wash step
may involve pushing or forcing a fluid solution over or through the
membrane (e.g., a buffer). The volume of wash solution may be from
about 10 .mu.L to about 50 mL. For example, the volume of wash
solution may be about 10 .mu.L, about 50 .mu.L, about 100 .mu.L,
about 200 .mu.L, about 300 .mu.L, about 400 .mu.L, about 500 .mu.L,
about 600 .mu.L, about 700 .mu.L, about 800 .mu.L, about 900 .mu.L,
about 1 mL, about 5 mL, about 10 mL, about 15 mL, about 20 mL,
about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL,
about 50 mL or greater than 50 mL. In other cases, the wash step
may involve pushing or forcing air over or through the membrane.
This step may be advantageous in decreasing the volume of sample
buffer that is carried over into the lysis buffer. The volume of
air wash may be from about 0.1 .mu.L to about 100 L, or about 10
.mu.L to about 50 mL. For example, the volume of air wash may be
about 10 about 50 about 100 about 200 about 300 about 400 about 500
about 600 about 700 about 800 about 900 about 1 mL, about 5 mL,
about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL,
about 35 mL, about 40 mL, about 45 mL, about 50 mL or greater than
50 mL. In some cases, an air wash volume of about 1-5 mL may be
preferred, For example an air wash may be have a volume of about
1.5 mL. In cases where an air wash is used the subsequent liquid
wash may be more effective and/or the final eluted sample may be
cleaner than if no air wash were used. In some cases, the wash step
involves both a fluid wash step and an air wash step, performed in
any order. In some cases, the wash solution comprises bovine serum
albumin and/or a detergent. In some cases, the wash solution
comprises about 0.1% to 5% bovine serum albumin. In some cases, the
wash solution comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%,
1.5%, 2%, 2.5%, 3%, 4%, or 5% bovine serum albumin. In some cases,
the wash solution comprises about 0.1% to 20% detergent. In some
cases, the wash solution comprises about 1%, 2%, 3%, 4%, 5%, 6%,
7%, 8%, 9%, or 10% detergent. In some cases, the detergent is
Tween-20. In some embodiments, the bovine serum albumin and/or
detergent increase the viscosity of the wash solution in manner
which increases the surface area of the filter contacted with the
wash solution during a wash step as compared to a wash solution
lacking one or both of bovine serum albumin and detergent.
[0081] After the membrane is washed, the biological cells or
entities captured on the membrane may be lysed or otherwise
disrupted so as to release a plurality of nucleic acid molecules
contained therein. The methods and devices of this disclosure may
use chemical, enzymatic and/or thermal methods to lyse the sample.
In some embodiments the methods and devices of this disclosure do
not use ultrasound to lyse the sample. In some cases, the cells may
be lysed by heating the sample. For example the sample may be
heated to greater than about 90.degree. C. for longer than about 10
seconds. In some examples heating the sample to about 95.degree. C.
for about 20 seconds is seen to be sufficient to lyse the
sample.
[0082] In some cases, lysis involves flowing a lysis buffer over
the biological cells or entities captured on the membrane. In some
cases, the lysis buffer is flowed through the filter membrane. In
other cases, the lysis buffer is back-flowed through the filter
membrane. The lysis buffer may be osmotically imbalanced so as to
force fluid into the cells to rupture the cell membranes. In some
cases, the lysis buffer may include one or more surfactants or
detergents. Non-limiting examples of surfactants or detergents that
may be used include: nonionic surfactants including polyoxyethylene
glycol alkyl ethers (sold as Brij.RTM. series detergents including
Brij.RTM. 58, Brij.RTM. 52, Brij.RTM. L4 and Brij.RTM. L23),
octaethylene glycol monododecyl ether, pentaethylene glycol
monododecyl ether, polyoxypropylene glycol alkyl ethers, glucoside
alkyl ethers (e.g., decyl glucoside, lauryl glucoside, octyl
glucoside), polyoxyethylene glycol octylphenol ethers (e.g., Triton
X-100), polyoxyethylene glycol alkylphenol ethers (e.g.,
nonoxynol-9), glycerol alkyl esters (e.g., glyceryl laurate),
polyoxyethylene glycol sorbitan alkyl esters (e.g., polyoxyethylene
glycol (20) sorbitan monolaurate, polyoxyethylene glycol (40)
sorbitan monolaurate, polyoxyethylene glycol (20) sorbitan
monopalmitate, polyoxyethylene glycol (20) sorbitan monostearate,
polyoxyethylene glycol (4) sorbitan monostearate, polyoxyethylene
glycol (20) sorbitan tristearate, polyoxyethylene glycol (20)
sorbitan monooleate)), sorbitan alkyl esters (e.g., sorbitan
monolaurate, sorbitan monopalmitate, sorbitan monostearate,
sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate,
sorbitan isostearate), cocamide monoethanolamine, cocamide
diethanolamine, dodecyldimethylamine oxide, poloxamers including
those sold under the Pluronic.RTM., Synperonic.RTM. and
Kolliphor.RTM. tradenames, and polyethoxylated tallow amine (POEA);
anionic surfactants including ammonium lauryl sulfate, ammonium
perfluorononanoate, docusate, perfluorobutanesulfonic acid,
perfluorononanoic acid, perfluorooctanesulfonic acid,
perfluorooctanoic acid, potassium lauryl sulfate, sodium alkyl
sulfate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate,
sodium laurate, sodium lauryl ether sulfate, sodium lauroyl
sarcosinate, sodium myreth sulfate, sodium pareth sulfate, sodium
stearate; cationic surfactants including benzalkonium chloride,
benzethonium chloride, bronidox, cetrimonium bromide, cetrimonium
chloride, distearyldimethylammonium chloride, lauryl methyl
gluceth-10 hydroxypropyl dimonium chloride, octenidine
dihydrochloride, olaflur, and tetramethylammonium hydroxide; and
Zwitterionic surfactants including CHAPS detergent, cocamidopropyl
betaine, cocamidopropyl hydroxysultaine,
dipalmitoylphosphatidylcholine, lecithin, hydroxysultaine, and
sodium lauroamphoacetate.
[0083] In some cases, the lysis buffer may contain an antifoaming
agent for preventing or minimizing foaming. Non-limiting examples
of antifoaming agents include Antifoam SE-15, Antifoam 204,
Antifoam Y-30. In some cases, the lysis buffer may contain a
preservative, for example an antimicrobial agent. Non-limiting
examples of antimicrobials may include ProClin.TM. 150, ProClin.TM.
200, ProClin.TM. 300, and ProClin.TM. 950.
[0084] In cases where the desired nucleic acid molecules are RNA,
the lysis buffer may include one or more agents that prevent
degradation of the RNA, such as, for example, an RNAse inhibitor.
The volume of lysis buffer flowed over the membrane can be from
about 10 .mu.L to about 50 mL. For example, the volume of lysis
buffer may be about 10 .mu.L, about 50 .mu.L, about 100 .mu.L,
about 200 .mu.L, about 300 .mu.L, about 400 .mu.L, about 500 .mu.L,
about 600 .mu.L, about 700 .mu.L, about 800 .mu.L, about 900 .mu.L,
about 1 mL, about 5 mL, about 10 mL, about 15 mL, about 20 mL,
about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL,
about 50 mL or greater than 50 mL.
[0085] In some cases, the lysis buffer contains one or more
enzymes. In some cases, the one or more enzymes comprise Proteinase
K. Proteinase K may be present in the lysis buffer at a
concentration of about 0.001 mg/mL to about 10 mg/mL. For example,
the concentration of proteinase K in the lysis buffer may be about
0.001 mg/mL, about 0.005 mg/mL, about 0.01 mg/mL, about 0.05 mg/mL,
about 0.1 mg/mL, about 0.5 mg/mL, about 1 mg/mL, about 2 mg/mL,
about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7
mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL or greater than
about 10 mg/mL. In some cases, the one or more enzymes comprise
lysozyme to process gram-positive organisms. Lysozyme may be
present in the lysis buffer at a concentration of about 0.001 mg/mL
to about 10 mg/mL. For example, the concentration of lysozyme in
the lysis buffer may be about 0.001 mg/mL, about 0.005 mg/mL, about
0.01 mg/mL, about 0.05 mg/mL, about 0.1 mg/mL, about 0.5 mg/mL,
about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5
mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL,
about 10 mg/mL or greater than about 10 mg/mL. In some cases, the
one or more enzymes comprise zymolyase to process yeast. Zymolase
may be present in the lysis buffer at a concentration of about
0.001 mg/mL to about 10 mg/mL. For example, the concentration of
zymolase in the lysis buffer may be about 0.001 mg/mL, about 0.005
mg/mL, about 0.01 mg/mL, about 0.05 mg/mL, about 0.1 mg/mL, about
0.5 mg/mL, about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4
mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL,
about 9 mg/mL, about 10 mg/mL or greater than about 10 mg/mL.
Additional enzymes that may be used include, without limitation,
lyticase, chitinase or gluculase, for e.g., the extraction of
nucleic acids from yeast. In some examples, if more than one lysis
enzyme is used, the enzymes may be added in sequence. For example,
lysozyme may be added first, followed by an incubation period, and
subsequently followed by addition of proteinase K and an additional
incubation period. In some cases, the lysis buffer does not contain
any enzymes.
[0086] In some aspects, the methods may involve one or more
incubation steps. The one or more incubation steps may be performed
in the lysis buffer in order to ensure complete lysis or disruption
of the biological cell or entity and/or to destroy any inhibitory
protein that may be present. The incubation step may involve
holding the biological cell or entity in the lysis buffer for a
period of time. In some cases, the incubation step involves holding
the biological cell or entity in the lysis buffer for a period of
time at a specified temperature. In a non-limiting example, the
biological cell or entity is incubated in the lysis buffer from
about 0.01 seconds to about 48 hours. For example, the biological
cell or entity is incubated in the lysis buffer from about 0.01
seconds, about 0.05 seconds, about 1 second, about 10 seconds,
about 30 seconds, about 1 minute, about 5 minutes, about 10
minutes, about 30 minutes, about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about
12 hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, about 24
hours, about 48 hours, or greater than 48 hours. In some examples,
the biological cell or entity is incubated in the lysis buffer at a
specified temperature, for example, from about 4.degree. C. to
about 75.degree. C. For example, the biological cell or entity is
incubated in the lysis buffer at a temperature of about 4.degree.
C., about 10.degree. C., about 15.degree. C., about 20.degree. C.,
about 25.degree. C., about 30.degree. C., about 40.degree. C.,
about 45.degree. C., about 50.degree. C., about 55.degree. C.,
about 60.degree. C., about 65.degree. C., about 70.degree. C.,
about 75.degree. C. or greater than 75.degree. C. Generally, the
temperature conditions will be selected so as to promote disruption
of the biological cell or entity. For example, if the lysis buffer
contains an enzyme (e.g., Proteinase K), the temperature may be
selected such that the enzyme retains catalytic activity. In some
cases, the temperature may be selected for optimal catalytic
activity of the lysis enzyme. The temperature may also be selected
to neutralize any inhibitory proteins within the sample, but should
not destroy or disrupt the integrity of the nucleic acid molecules
released therefrom. In some cases, the lysis buffer does not
contain any enzymes.
[0087] The presence of one or more components (e.g., Proteinase K)
in the lysis buffer may affect or interfere with downstream
applications. In some cases, an additional incubation step may be
performed to, for example, destroy or inactivate the one or more
interfering components (e.g., Proteinase K) used in the lysis step.
The subsequent incubation step may be from about 0.01 seconds to
about 48 hours. For example, the biological cell or entity is
incubated in the lysis buffer from about 0.01 seconds, about 0.05
seconds, about 1 second, about 10 seconds, about 30 seconds, about
1 minute, about 5 minutes, about 10 minutes, about 30 minutes,
about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5
hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,
about 10 hours, about 11 hours, about 12 hours, about 13 hours,
about 14 hours, about 15 hours, about 16 hours, about 17 hours,
about 18 hours, about 19 hours, about 20 hours, about 21 hours,
about 22 hours, about 23 hours, about 24 hours, about 48 hours, or
greater than 48 hours. In some examples, the additional incubation
step may occur at a temperature between about 57.degree. C. and
about 100.degree. C. For example, the additional incubation step
may occur at a temperature of about 57.degree. C., about 58.degree.
C., about 59.degree. C., about 60.degree. C., about 61.degree. C.,
about 62.degree. C., about 63.degree. C., about 64.degree. C.,
about 65.degree. C., about 66.degree. C., about 67.degree. C.,
about 68.degree. C., about 69.degree. C., about 70.degree. C.,
about 71.degree. C., about 72.degree. C., about 73.degree. C.,
about 74.degree. C., about 75.degree. C., about 76.degree. C.,
about 77.degree. C., about 78.degree. C., about 79.degree. C.,
about 80.degree. C., about 81.degree. C., about 82.degree. C.,
about 83.degree. C., about 84.degree. C., about 85.degree. C.,
about 86.degree. C., about 87.degree. C., about 88.degree. C.,
about 89.degree. C., about 90.degree. C., about 91.degree. C.,
about 92.degree. C., about 93.degree. C., about 94.degree. C.,
about 95.degree. C., about 96.degree. C., about 97.degree. C.,
about 98.degree. C., about 99.degree. C., about 100.degree. C. or
greater than 100.degree. C.
[0088] In some aspects, the extracted nucleic acids may be utilized
at this stage for any downstream processes, without any
purification steps. In some cases, the extracted nucleic acid
molecules may be used in one or more amplification reactions. For
example, the extracted nucleic acid molecules may be used in one or
more polymerase chain reactions (PCR). Any known method of PCR may
be performed using the extracted nucleic acid molecules provided
herein.
[0089] In some cases, when RNA is extracted, the RNA may be reverse
transcribed (i.e., using a reverse transcriptase) prior to
performing the downstream application. Briefly this may occur as in
the diagram in FIG. 28, the sample is processed in a pre-sample
prep stage which may include concentration, purification and lysis
of the sample, the sample then moves to a RT-PCR step in which RNA
molecules are reverse transcribed to DNA molecules, these move to a
mixing compartment and thence to a PCR module and a detection
module. Optionally this may occur as in FIG. 34 which includes an
additional step between the pre-sample prep stage and the RT-PCR
step in which the sample is mixed with reagents for performing the
reverse transcriptase reaction. In some embodiments the steps of
reverse transcription and PCR may occur in the same module, in this
case the amplification module. Extracted RNA molecules may be
incubated with one or more reverse transcriptase enzymes at a
suitable temperature for reverse transcription to occur. The
reverse transcriptase enzyme may be provided alone or with a buffer
suitable for the reverse transcriptase reaction. The reverse
transcriptase may be provided with a concentrated buffer designed
to adjust the conditions of the extracted nucleic acid solution. In
other cases no additional components are provided and the lysis
buffer is suitable for reverse transcriptase. The incubation step
may involve holding the biological cell or entity in the buffer for
a period of time. In some cases, the incubation step involves
holding the RNA molecule in the buffer for a period of time at a
specified temperature. In a non-limiting example, the RNA molecule
is incubated in the buffer from about 0.01 seconds to about 48
hours. For example, the RNA molecule is incubated in the buffer
from about 0.01 seconds, about 0.05 seconds, about 1 second, about
10 seconds, about 30 seconds, about 1 minute, about 5 minutes,
about 10 minutes, about 30 minutes, about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 10 hours, about 11
hours, about 12 hours, about 13 hours, about 14 hours, about 15
hours, about 16 hours, about 17 hours, about 18 hours, about 19
hours, about 20 hours, about 21 hours, about 22 hours, about 23
hours, about 24 hours, about 48 hours, or greater than 48 hours. In
some examples, the RNA molecule is incubated in the buffer at a
specified temperature, for example, from about 4.degree. C. to
about 75.degree. C. For example, the RNA molecule is incubated in
the buffer at a temperature of about 4.degree. C., about 10.degree.
C., about 15.degree. C., about 20.degree. C., about 25.degree. C.,
about 30.degree. C., about 37, about 40.degree. C., about
45.degree. C., about 50.degree. C., about 55.degree. C., about
60.degree. C., about 65.degree. C., about 70.degree. C., about
75.degree. C. or greater than 75.degree. C. Generally, the
temperature conditions will be selected so as to promote activity
of the reverse transcriptase enzyme. An example of a temperature
profile for the reverse transcription reaction and inactivation
step is shown by FIG. 29. The temperature of the RNA containing
sample is heated to a temperature suitable for the RT reaction
(T.sub.RT). The temperature T.sub.RT is reached by a first time
(t1) and maintained for a period of time suitable to complete the
reaction (t.sub.1 to t.sub.2). In the next stage from time t.sub.2
to time t.sub.3 the sample is heated to a temperature sufficient to
inactivate the RT enzyme (T.sub.inact). The sample is maintained at
this temperature from time t.sub.3 to time t.sub.4, which provides
a suitable amount of time to inactive the RT enzyme at a
temperature of T.sub.inact.
[0090] The presence of the reverse transcriptase in the buffer may
affect or interfere with downstream applications. In some cases, an
additional incubation step may be performed to, for example,
destroy or inactivate the reverse transcriptase enzyme. The
subsequent incubation step may be from about 0.01 seconds to about
48 hours. For example, the mixture of RNA and DNA molecules
produced by the reverse transcriptase step is incubated from about
0.01 seconds, about 0.05 seconds, about 1 second, about 10 seconds,
about 30 seconds, about 1 minute, about 5 minutes, about 10
minutes, about 30 minutes, about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about
12 hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, about 24
hours, about 48 hours, or greater than 48 hours. In some examples,
the additional incubation step may occur at a temperature between
about 57.degree. C. and about 100.degree. C. For example, the
additional incubation step may occur at a temperature of about
57.degree. C., about 58.degree. C., about 59.degree. C., about
60.degree. C., about 61.degree. C., about 62.degree. C., about
63.degree. C., about 64.degree. C., about 65.degree. C., about
66.degree. C., about 67.degree. C., about 68.degree. C., about
69.degree. C., about 70.degree. C., about 71.degree. C., about
72.degree. C., about 73.degree. C., about 74.degree. C., about
75.degree. C., about 76.degree. C., about 77.degree. C., about
78.degree. C., about 79.degree. C., about 80.degree. C., about
81.degree. C., about 82.degree. C., about 83.degree. C., about
84.degree. C., about 85.degree. C., about 86.degree. C., about
87.degree. C., about 88.degree. C., about 89.degree. C., about
90.degree. C., about 91.degree. C., about 92.degree. C., about
93.degree. C., about 94.degree. C., about 95.degree. C., about
96.degree. C., about 97.degree. C., about 98.degree. C., about
99.degree. C., about 100.degree. C. or greater than 100.degree.
C.
[0091] Biological Samples
[0092] In some cases, the biological sample can be a tissue sample.
In some cases, the tissue sample is a blood sample. In some cases,
the biological sample comprises a bodily fluid taken from a
subject. In some cases, the bodily fluid comprises one or more
cells comprising nucleic acids. In some cases, the one or more
cells comprise one or more microbial cells, including, but not
limited to, bacteria, archaebacteria, protists, and fungi. In some
cases, the biological sample includes one or more virus particles.
In some cases, the biological sample includes one or more RNA based
virus particles. In some cases, the biological sample comprises one
or more microbes that causes a sexually-transmitted disease. A
sample may comprise a sample from a subject, such as whole blood;
blood products; red blood cells; white blood cells; buffy coat;
swabs; urine; sputum; saliva; semen; lymphatic fluid; endolymph;
perilymph; gastric juice; bile; mucus; sebum; sweat; tears; vaginal
secretion; vomit; feces; breast milk; cerumen; amniotic fluid;
cerebrospinal fluid; peritoneal effusions; pleural effusions;
biopsy samples; fluid from cysts; synovial fluid; vitreous humor;
aqueous humor; bursa fluid; eye washes; eye aspirates; plasma;
serum; pulmonary lavage; lung aspirates; animal, including human,
tissues, including but not limited to, liver, spleen, kidney, lung,
intestine, brain, heart, muscle, pancreas, cell cultures, as well
as lysates, extracts, or materials and fractions obtained from the
samples described above or any cells and microorganisms and viruses
that may be present on or in a sample. A sample may comprise cells
of a primary culture or a cell line. Examples of cell lines
include, but are not limited to 293-T human kidney cells, A2870
human ovary cells, A431 human epithelium, B35 rat neuroblastoma
cells, BHK-21 hamster kidney cells, BR293 human breast cells, CHO
Chinese hamster ovary cells, CORL23 human lung cells, HeLa cells,
or Jurkat cells. The sample may comprise a homogeneous or mixed
population of microbes, including one or more of viruses, bacteria,
protists, monerans, chromalveolata, archaea, or fungi. The
biological sample can be a urine sample, a vaginal swab, a cervical
swab, an anal swab, or a cheek swab. The biological sample can be
obtained from a hospital, laboratory, clinical or medical
laboratory. The sample can be obtained from a subject.
[0093] Non-limiting examples of sample sources include
environmental sources, industrial sources, one or more subjects,
and one or more populations of microbes. Examples of environmental
sources include, but are not limited to agricultural fields, lakes,
rivers, water reservoirs, air vents, walls, roofs, soil samples,
plants, and swimming pools. Examples of industrial sources include,
but are not limited to clean rooms, hospitals, food processing
areas, food production areas, food stuffs, medical laboratories,
pharmacies, and pharmaceutical compounding centers. Examples of
subjects from which polynucleotides may be isolated include
multicellular organisms, such as fish, amphibians, reptiles, birds,
and mammals. Examples of mammals include primates (e.g., apes,
monkeys, gorillas), rodents (e.g., mice, rats), cows, pigs, sheep,
horses, dogs, cats, or rabbits. In some examples, the mammal is a
human. In some cases, the sample is from an individual subject.
[0094] In some cases, the biological sample is provided in a sample
buffer. In some cases, the sample buffer comprises bovine serum
albumin and/or a detergent. In some cases, the sample buffer
comprises about 0.1% to 5% bovine serum albumin. In some cases, the
sample buffer comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%,
1.5%, 2%, 2.5%, 3%, 4%, or 5% bovine serum albumin. In some cases,
the sample buffer comprises about 0.1% to 20% detergent. In some
cases, the sample buffer comprises about 1%, 2%, 3%, 4%, 5%, 6%,
7%, 8%, 9%, or 10% detergent. In some cases, the detergent is
Tween-20. The choice of sample buffer to be used may depend on the
intended method. For example the choice of sample buffer may
different when a wash step will be used to when a wash step is not
used. If a wash step will not be used then the sample buffer may be
a buffer suitable for lysis and subsequent PCR reactions.
[0095] Some commercial collection mediums or sample buffers contain
chemicals for the preservation of microorganisms for future growth,
or chemicals that lyse target organisms such as guanidinium
thiocyanate. As such, these collection media are inhibitory to DNA
polymerase and must be washed from a sample before PCR via
filtration or similar process. The methods described herein may not
require the target organism to be kept in a viable state, or for
the sample buffer to be able to lyse the cells. Some components
which may be found in a sample buffer suitable for use with the
methods and devices of this disclosure include: Tris HCL, Tween-80,
BSA, Proclin and Antifoam SE-15. In one embodiment a sample buffer
may have a composition of: 50 mM Tris pH 8.4, Tween-80, 2% (w/v),
BSA, 0.25% (w/v), Proclin 300, 0.03% (w/v), and Antifoam SE-15,
0.002% (v/v) made up in purified water.
[0096] Tris HCL is a common buffer for PCR. When it is heated
during thermocycling, the pH may drop, for example a Tris buffer
with pH of 8.4 at a temperature of 25.degree. C. may drop to a pH
of about .about.7.4 when heated to about 95.degree. C. The range of
concentrations could be from 0.1 mM to 1 M. The pH range could be
from 6 to 10. Any other PCR compatible buffer could be used, for
example HEPES.
[0097] Tween-80 is a nonionic surfactant and emulsifier that may
help to elute target organisms off of a swab. The range of
concentrations could be from 0.01% (w/v) to 20% (w/v). Any other
PCR compatible surfactant and/or emulsifier could be used.
[0098] Proclin 300 is a broad spectrum antimicrobial used as a
preservative to ensure a long shelf life of the collection media.
It could be used from 0.01% (w/v) to 0.1% (w/v). Many other
antimicrobials are known in the art and could be used in a sample
buffer.
[0099] Antifoam SE-15 is present to reduce foaming during
manufacturing and fluidic movement through the device. It could be
used from 0.001% (v/v) to 1% (v/v). Any other antifoam agent could
also be used, for example Antifoam 204, Antifoam A, Antifoam B,
Antifoam C, or Antifoam Y-30.
[0100] The devices and methods provided herein may be utilized to
prepare nucleic acids for downstream applications. The downstream
applications may be utilized to, e.g., detect the presence or
absence of a nucleic acid sequence present in the sample. In some
instances, the devices and methods can be utilized to detect the
presence or absence of one or more microbes in a biological sample.
In some cases, the one or more microbes are pathogens (i.e.,
disease-causative). In some cases, the one or more microbes are
infectious. In some cases, the one or more microbes cause disease
in a subject. In some cases, the disease is a sexually transmitted
disease.
[0101] In some aspects, the devices and methods can be utilized to
detect the presence or absence of nucleic acids associated with one
or more bacterial cells in the biological sample. In some cases,
one or more bacterial cells are pathogens. In some cases, the one
or more bacterial cells are infectious. Non-limiting examples of
bacterial pathogens that can be detected include Mycobacteria (e.g.
M. tuberculosis, M. bovis, M. avium, M. leprae, and M. africanum),
rickettsia, mycoplasma, chlamydia, and legionella. Some examples of
bacterial infections include, but are not limited to, infections
caused by Gram positive bacillus (e.g., Listeria, Bacillus such as
Bacillus anthracis, Erysipelothrix species), Gram negative bacillus
(e.g., Bartonella, Brucella, Campylobacter, Enterobacter,
Escherichia, Francisella, Hemophilus, Klebsiella, Morganella,
Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella,
Vibrio and Yersinia species), spirochete bacteria (e.g., Borrelia
species including Borrelia burgdorferi that causes Lyme disease),
anaerobic bacteria (e.g., Actinomyces and Clostridium species),
Gram positive and negative coccal bacteria, Enterococcus species,
Streptococcus species, Pneumococcus species, Staphylococcus
species, and Neisseria species. Specific examples of infectious
bacteria include, but are not limited to: Helicobacter pyloris,
Legionella pneumophilia, Mycobacterium tuberculosis, Mycobacterium
avium, Mycobacterium intracellulare, Mycobacterium kansaii,
Mycobacterium gordonae, Staphylococcus aureus, Neisseria
gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,
Streptococcus pyogenes (Group A Streptococcus), Streptococcus
agalactiae (Group B Streptococcus), Streptococcus viridans,
Streptococcus faecalis, Streptococcus bovis, Streptococcus
pneumoniae, Haemophilus influenzae, Bacillus antracis,
Erysipelothrix rhusiopathiae, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasteurella multocida,
Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema
pallidium, Treponema pertenue, Leptospira, Rickettsia, and
Actinomyces israelii, Acinetobacter, Bacillus, Bordetella,
Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila,
Clostridium, Corynebacterium, Enterococcus, Haemophilus,
Helicobacter, Mycobacterium, Mycoplasma, Stenotrophomonas,
Treponema, Vibrio, Yersinia, Acinetobacter baumanii, Bordetella
pertussis, Brucella abortus, Brucella canis, Brucella melitensis,
Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae,
Chlamydia trachomatis, Chlamydophila psittaci, Clostridium
botulinum, Clostridium difficile, Clostridium perfringens,
Corynebacterium diphtheriae, Enterobacter sazakii, Enterobacter
agglomerans, Enterobacter cloacae, Enterococcus faecalis,
Enterococcus faecium, Escherichia coli, Francisella tularensis,
Helicobacter pylori, Legionella pneumophila, Leptospira
interrogans, Mycobacterium leprae, Mycobacterium tuberculosis,
Mycobacterium ulcerans, Mycoplasma pneumoniae, Pseudomonas
aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella
typhimurium, Salmonella enterica, Shigella sonnei, Staphylococcus
epidermidis, Staphylococcus saprophyticus, Stenotrophomonas
maltophilia, Vibrio cholerae, Yersinia pestis, and the like. In
some instances, the infectious bacteria is Neisseria gonorrhoeae or
Chlamydia trachomatis.
[0102] In some aspects, the devices and methods can be utilized to
detect the presence or absence of nucleic acids associated with one
or more viruses in the biological sample. Non-limiting examples of
types of viruses include double stranded DNA viruses, single
stranded DNA viruses, double stranded RNA viruses, or single
stranded RNA viruses. Single stranded RNA viruses may replicate
directly or may include a DNA intermediate in their lifecycle. DNA
viruses may replicate directly or through an RNA intermediate.
Non-limiting examples of viruses include the herpes virus (e.g.,
human cytomegalomous virus (HCMV), herpes simplex virus 1 (HSV-1),
herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV),
Epstein-Barr virus), influenza A virus and Hepatitis C virus (HCV)
or a picornavirus such as Coxsackievirus B3 (CVB3). Other viruses
may include, but are not limited to, the hepatitis B virus, HIV,
poxvirus, hepadavirus, retrovirus, and RNA viruses such as
flavivirus, togavirus, coronavirus, Hepatitis D virus,
orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filo virus,
Adenovirus, Human herpesvirus, type 8, Human papillomavirus, BK
virus, JC virus, Smallpox, Hepatitis B virus, Human bocavirus,
Parvovirus B19, Human astrovirus, Norwalk virus, coxsackievirus,
hepatitis A virus, poliovirus, rhinovirus, Severe acute respiratory
syndrome virus, Hepatitis C virus, yellow fever virus, dengue
virus, West Nile virus, Rubella virus, Hepatitis E virus, and Human
immunodeficiency virus (HIV). In some cases, the virus is an
enveloped virus. Examples include, but are not limited to, viruses
that are members of the hepadnavirus family, herpesvirus family,
iridovirus family, poxvirus family, flavivirus family, togavirus
family, retrovirus family, coronavirus family, filovirus family,
rhabdovirus family, bunyavirus family, orthomyxovirus family,
paramyxovirus family, and arenavirus family. Other examples
include, but are not limited to, Hepadnavirus hepatitis B virus
(HBV), woodchuck hepatitis virus, ground squirrel (Hepadnaviridae)
hepatitis virus, duck hepatitis B virus, heron hepatitis B virus,
Herpesvirus herpes simplex virus (HSV) types 1 and 2,
varicella-zoster virus, cytomegalovirus (CMV), human
cytomegalovirus (HCMV), mouse cytomegalovirus (MCMV), guinea pig
cytomegalovirus (GPCMV), Epstein-Barr virus (EBV), human herpes
virus 6 (HHV variants A and B), human herpes virus 7 (HHV-7), human
herpes virus 8 (HHV-8), Kaposi's sarcoma-associated herpes virus
(KSHV), B virus Poxvirus vaccinia virus, variola virus, smallpox
virus, monkeypox virus, cowpox virus, camelpox virus, ectromelia
virus, mousepox virus, rabbitpox viruses, raccoonpox viruses,
molluscum contagiosum virus, orf virus, milker's nodes virus, bovin
papullar stomatitis virus, sheeppox virus, goatpox virus, lumpy
skin disease virus, fowlpox virus, canarypox virus, pigeonpox
virus, sparrowpox virus, myxoma virus, hare fibroma virus, rabbit
fibroma virus, squirrel fibroma viruses, swinepox virus, tanapox
virus, Yabapox virus, Flavivirus dengue virus, hepatitis C virus
(HCV), GB hepatitis viruses (GBV-A, GBV-B and GBV-C), West Nile
virus, yellow fever virus, St. Louis encephalitis virus, Japanese
encephalitis virus, Powassan virus, tick-borne encephalitis virus,
Kyasanur Forest disease virus, Togavirus, Venezuelan equine
encephalitis (VEE) virus, chikungunya virus, Ross River virus,
Mayaro virus, Sindbis virus, rubella virus, Retrovirus human
immunodeficiency virus (HIV) types 1 and 2, human T cell leukemia
virus (HTLV) types 1, 2, and 5, mouse mammary tumor virus (MMTV),
Rous sarcoma virus (RSV), lentiviruses, Coronavirus, severe acute
respiratory syndrome (SARS) virus, Filovirus Ebola virus, Marburg
virus, Metapneumoviruses (MPV) such as human metapneumovirus
(HMPV), Rhabdovirus rabies virus, vesicular stomatitis virus,
Bunyavirus, Crimean-Congo hemorrhagic fever virus, Rift Valley
fever virus, La Crosse virus, Hantaan virus, Orthomyxovirus,
influenza virus (types A, B, and C), Paramyxovirus, parainfluenza
virus (PIV types 1, 2 and 3), respiratory syncytial virus (types A
and B), measles virus, mumps virus, Arenavirus, lymphocytic
choriomeningitis virus, Junin virus, Machupo virus, Guanarito
virus, Lassa virus, Ampari virus, Flexal virus, Ippy virus, Mobala
virus, Mopeia virus, Latino virus, Parana virus, Pichinde virus,
Punta toro virus (PTV), Tacaribe virus and Tamiami virus. In some
embodiments, the virus is a non-enveloped virus, examples of which
include, but are not limited to, viruses that are members of the
parvovirus family, circovirus family, polyoma virus family,
papillomavirus family, adenovirus family, iridovirus family,
reovirus family, birnavirus family, calicivirus family, and
picornavirus family. Specific examples include, but are not limited
to, canine parvovirus, parvovirus B19, porcine circovirus type 1
and 2, BFDV (Beak and Feather Disease virus, chicken anaemia virus,
Polyomavirus, simian virus 40 (SV40), JC virus, BK virus,
Budgerigar fledgling disease virus, human papillomavirus, bovine
papillomavirus (BPV) type 1, cotton tail rabbit papillomavirus,
human adenovirus (HAdV-A, HAdV-B, HAdV-C, HAdV-D, HAdV-E, and
HAdV-F), fowl adenovirus A, bovine adenovirus D, frog adenovirus,
Reovirus, human orbivirus, human coltivirus, mammalian
orthoreovirus, bluetongue virus, rotavirus A, rotaviruses (groups B
to G), Colorado tick fever virus, aquareovirus A, cypovirus 1, Fiji
disease virus, rice dwarf virus, rice ragged stunt virus,
idnoreovirus 1, mycoreovirus 1, Birnavirus, bursal disease virus,
pancreatic necrosis virus, Calicivirus, swine vesicular exanthema
virus, rabbit hemorrhagic disease virus, Norwalk virus, Sapporo
virus, Picornavirus, human polioviruses (1-3), human
coxsackieviruses Al-22, 24 (CA1-22 and CA24, CA23 (echovirus 9)),
human coxsackieviruses (Bl-6 (CBl-6)), human echoviruses 1-7, 9,
11-27, 29-33, vilyuish virus, simian enteroviruses 1-18 (SEV1-18),
porcine enteroviruses 1-11 (PEV1-11), bovine enteroviruses 1-2
(BEV1-2), hepatitis A virus, rhinoviruses, hepatoviruses, cardio
viruses, aphthoviruses and echoviruses. The virus may be phage.
Examples of phages include, but are not limited to T4, T5, .lamda.
phage, T7 phage, G4, P1, .phi.6, Thermoproteus tenax virus 1, M13,
MS2, Q.beta., .phi.X174, .PHI.29, PZA, .PHI.15, BS32, B103, M2Y
(M2), Nf, GA-1, FWLBc1, FWLBc2, FWLLm3, B4. In some cases, the
virus is selected from a member of the Flaviviridae family (e.g., a
member of the Flavivirus, Pestivirus, and Hepacivirus genera),
which includes the hepatitis C virus, Yellow fever virus;
Tick-borne viruses, such as the Gadgets Gully virus, Kadam virus,
Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever
virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne
encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill
virus and the Negishi virus; seabird tick-borne viruses, such as
the Meaban virus, Saumarez Reef virus, and the Tyuleniy virus;
mosquito-borne viruses, such as the Aroa virus, dengue virus,
Kedougou virus, Cacipacore virus, Koutango virus, Japanese
encephalitis virus, Murray Valley encephalitis virus, St. Louis
encephalitis virus, Usutu virus, West Nile virus, Yaounde virus,
Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey
meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika
virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus,
Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus,
yellow fever virus; and viruses with no known arthropod vector,
such as the Entebbe bat virus, Yokose virus, Apoi virus, Cowbone
Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San
Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat
virus, Montana myotis leukoencephalitis virus, Phnom Penh bat
virus, Rio Bravo virus, Tamana bat virus, and the Cell fusing agent
virus. In some cases, the virus is selected from a member of the
Arenaviridae family, which includes the Ippy virus, Lassa virus
(e.g., the Josiah, LP, or GA391 strain), lymphocytic
choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari
virus, Flexal virus, Guanarito virus, Junin virus, Latino virus,
Machupo virus, Oliveros virus, Parana virus, Pichinde virus,
Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus,
Whitewater Arroyo virus, Chapare virus, and Lujo virus. In some
cases, the virus is selected from a member of the Bunyaviridae
family (e.g., a member of the Hantavirus, Nairovirus,
Orthobunyavirus, and Phlebovirus genera), which includes the
Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus,
Rift Valley fever virus, La Crosse virus, Punta Toro virus (PTV),
California encephalitis virus, and Crimean-Congo hemorrhagic fever
(CCHF) virus. In some cases, the virus is selected from a member of
the Filoviridae family, which includes the Ebola virus (e.g., the
Zaire, Sudan, Ivory Coast, Reston, and Uganda strains) and the
Marburg virus (e.g., the Angola, Ci67, Musoke, Popp, Ravn and Lake
Victoria strains); a member of the Togaviridae family (e.g., a
member of the Alphavirus genus), which includes the Venezuelan
equine encephalitis virus (VEE), Eastern equine encephalitis virus
(EEE), Western equine encephalitis virus (WEE), Sindbis virus,
rubella virus, Semliki Forest virus, Ross River virus, Barmah
Forest virus, O'nyong'nyong virus, and the chikungunya virus; a
member of the Poxyiridae family (e.g., a member of the
Orthopoxvirus genus), which includes the smallpox virus, monkeypox
virus, and vaccinia virus; a member of the Herpesviridae family,
which includes the herpes simplex virus (HSV; types 1, 2, and 6),
human herpes virus (e.g., types 7 and 8), cytomegalovirus (CMV),
Epstein-Barr virus (EBV), Varicella-Zoster virus, and Kaposi's
sarcoma associated-herpesvirus (KSHV); a member of the
Orthomyxoviridae family, which includes the influenza virus (A, B,
and C), such as the H5N1 avian influenza virus or H1N1 swine flu; a
member of the Coronaviridae family, which includes the severe acute
respiratory syndrome (SARS) virus; a member of the Rhabdoviridae
family, which includes the rabies virus and vesicular stomatitis
virus (VSV); a member of the Paramyxoviridae family, which includes
the human respiratory syncytial virus (RSV), Newcastle disease
virus, hendravirus, nipahvirus, measles virus, rinderpest virus,
canine distemper virus, Sendai virus, human parainfluenza virus
(e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus; a member of
the Picornaviridae family, which includes the poliovirus, human
enterovirus (A, B, C, and D), hepatitis A virus, and the
coxsackievirus; a member of the Hepadnaviridae family, which
includes the hepatitis B virus; a member of the Papillamoviridae
family, which includes the human papilloma virus; a member of the
Parvoviridae family, which includes the adeno-associated virus; a
member of the Astroviridae family, which includes the astrovirus; a
member of the Polyomaviridae family, which includes the JC virus,
BK virus, and SV40 virus; a member of the Calciviridae family,
which includes the Norwalk virus; a member of the Reoviridae
family, which includes the rotavirus; and a member of the
Retroviridae family, which includes the human immunodeficiency
virus (HIV; e.g., types 1 and 2), and human T-lymphotropic virus
Types I and II (HTLV-1 and HTLV-2, respectively).
[0103] In some aspects, the devices and methods can be utilized to
detect the presence or absence of nucleic acids associated with one
or more fungi in the biological sample. Examples of infectious
fungal agents include, without limitation Aspergillus, Blastomyces,
Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides,
Sporothrix, and at least three genera of Zygomycetes. The above
fungi, as well as many other fungi, can cause disease in pets and
companion animals. The present teaching is inclusive of substrates
that contact animals directly or indirectly. Examples of organisms
that cause disease in animals include Malassezia furfur,
Epidermophyton floccosur, Trichophyton mentagrophytes, Trichophyton
rubrum, Trichophyton tonsurans, Trichophyton equinum, Dermatophilus
congolensis, Microsporum canis, Microsporu audouinii, Microsporum
gypseum, Malassezia ovale, Pseudallescheria, Scopulariopsis,
Scedosporium, and Candida albicans. Further examples of fungal
infectious agent include, but are not limited to, Aspergillus,
Blastomyces dermatitidis, Candida, Coccidioides immitis,
Cryptococcus neoformans, Histoplasma capsulatum var. capsulatum,
Paracoccidioides brasiliensis, Sporothrix schenckii, Zygomycetes
spp., Absidia corymbifera, Rhizomucor pusillus, or Rhizopus
arrhizus.
[0104] In some aspects, the devices and methods can be utilized to
detect the presence or absence of nucleic acids associated with one
or more parasites in the biological sample. Non-limiting examples
of parasites include Plasmodium, Leishmania, Babesia, Treponema,
Borrelia, Trypanosoma, Toxoplasma gondii, Plasmodium falciparum, P.
vivax, P. ovale, P. malariae, Trypanosoma spp., or Legionella spp.
In some cases, the parasite is Trichomonas vaginalis.
[0105] In some cases, the biological sample can be an environmental
sample comprising medium such as water, soil, air, and the like. In
some cases, the biological sample can be a forensic sample (e.g.,
hair, blood, semen, saliva, etc.). In some cases, the biological
sample can comprise an agent used in a bioterrorist attack (e.g.,
influenza, anthrax, smallpox).
[0106] In some aspects, the biological sample comprises an
infectious agent associated with a sexually-transmitted disease
(STD) or a sexually-transmitted infection (STI). Non-limiting
examples of STDs or STIs and associated infectious agents that may
be detected with the devices and methods provided herein may
include, Bacterial Vaginosis; Chlamydia (Chlamydia trachomatis);
Genital herpes (herpes virus); Gonorrhea (Neisseria gonorrhoeae);
Hepatitis B (Hepatitis B virus); Hepatitis C (Hepatitis C virus);
Genital Warts, Anal Warts, Cervical Cancer (Human Papillomavirus);
Lymphogranuloma venereum (Chlamydia trachomatis); Syphilis
(Treponema pallidum); Trichomoniasis (Trichomonas vaginalis); Yeast
infection (Candida); and Acquired Immunodeficiency Syndrome (Human
Immunodeficiency Virus).
[0107] Performance
[0108] In some cases, the devices and methods described herein may
demonstrate improved performance when compared with traditional
methods. For example, in some cases, the devices and methods may
result in the extraction and preparation of nucleic acid molecules
suitable for use in a polymerase chain reaction (PCR) in a shorter
period of time when compared with other methods. In some cases, the
devices and methods may result in the extraction and preparation of
nucleic acid molecules suitable for use in a PCR reaction in 20
minutes or less. For example, the extraction and preparation of
nucleic acid molecules as described herein may be achieved in about
20 minutes, 19 minutes, 18 minutes, 17 minutes, 16 minutes, 15
minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10
minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4
minutes, 3 minutes, 2 minutes, 1 minute or less than 1 minute. In
some cases, the extraction and preparation of nucleic acid
molecules as described herein is achieved in about 5 minutes or
less. In some cases, the method extracts nucleic acid molecules in
about 5 minutes or less at a quality sufficient to successfully run
a polymerase chain reaction (PCR).
[0109] A quality of extracted or prepared nucleic acid sufficient
to run a polymerase chain reaction refers to the quantity of
extracted or prepared nucleic acid, the purity of the nucleic acid
and the shearing of the nucleic acid (average length of nucleic
acid molecules). A sufficient quantity of nucleic acid may refer to
about 0.001, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, or 1 .mu.g. A sufficient quantity may also refer to the
concentration of the nucleic acid in the eluted liquid. The
concentration of the eluted nucleic acid may be about 0.001, 0.01,
0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 .mu.g/.mu.L.
The nucleic acid produced may comprise nucleic acid fragments with
an average length of at least about 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000 or more than 1000 base pairs.
[0110] A quality of extracted or prepared nucleic acid sufficient
to run a polymerase chain reaction may be a sample that produces at
least 70% efficiency as determined by a qPCR standard curve. The
efficiency of the PCR may be between 90-100%
(-3.6.gtoreq.slope.gtoreq.-3.3). Efficiency of qPCR may be
quantified by calculating the cycle difference between a sample and
10-fold dilution of the sample. For example if the efficiency is
100%, the Ct values of a 10 fold dilution of input DNA will be 3.3
cycles apart (there is a 2-fold change for each change in Ct).
[0111] In some cases, the nucleic acid sample extracted or prepared
using the devices and methods described herein have similar or
improved purity as compared to nucleic acid samples prepared using
other methods. The purity may be measured, for example, as a ratio
of the absorbance at 260 nm and 280 nm (e.g., A260/A280). For
example, a nucleic acid samples comprising DNA prepared using the
devices and methods may have a A260/A280 ratio of about 1.5, about
1.6, about 1.7, about 1.8, about 1.9, or about 2.0. In some cases,
the extracted or prepared nucleic acid molecules comprise DNA and
the DNA has an A260/A280 ratio of at least 1.5. In another example,
a nucleic acid sample comprising RNA prepared using the devices and
methods may have an A260/A280 ratio of about 1.7, about 1.8, about
1.9, about 2.0, about 2.1, or about 2.2. In some cases, the
extracted nucleic acid molecules comprise RNA and the RNA has an
A260/A280 ratio of at least 1.7.
[0112] Downstream processes such as polymerase chain reaction (PCR)
may be sensitive to certain molecules present in a sample. For
example, the presence of one or more lysis reagents (e.g.,
Proteinase K) may hinder or inhibit downstream processes. In some
cases, the nucleic acid molecules described herein are extracted
from the one or more biological cells or entities with a quality
that is sufficient to successfully perform one or more downstream
processes. In some cases, the extracted nucleic acid molecules may
be of a quality sufficient to successfully perform a PCR. For
example, the extracted nucleic acid molecules may be of a quality
sufficient to perform an amplification reaction on a target nucleic
acid molecule present in the extracted nucleic acid molecules to
generate amplified target nucleic molecules. In some cases, a
positive control may be used (e.g., a biological cell that is known
to be positive for the target molecule) to confirm that the
extraction process is performed successfully. The extracted nucleic
acid molecules described herein are generally substantially free of
molecules that inhibit downstream processes (e.g., Proteinase
K).
[0113] In some cases, the nucleic acid samples may have similar or
improved yields as compared to nucleic acid samples prepared using
other methods from the same amount of starting material. For
example, nucleic acid samples prepared using the methods and
devices described herein may have about 5%, about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 95%, about 99% or greater
yields than using other nucleic acid extraction methods from the
same amount of starting material.
[0114] Standard nucleic acid extraction methods may involve the use
of centrifuges and vacuums. In some cases, the methods and devices
herein do not involve the use of centrifuges or vacuums.
[0115] Devices
[0116] In some aspects, devices are provided for performing any of
the methods described herein. For example, FIG. 10 is a schematic
illustration of a molecular diagnostic test device 1000 (also
referred to as a "test device" or "device"), according to an
embodiment. The schematic illustration describes the primary
components of the test device 1000 as shown in FIG. 11. The test
device 1000 is an integrated device (i.e., the modules are
contained within a single housing) that is suitable for use within
a point-of-care setting (e.g., doctor's office, pharmacy or the
like), decentralized test facility, or at the user's home. In some
embodiments, the device 1000 can have a size, shape and/or weight
such that the device 1000 can be carried, held, used and/or
manipulated in a user's hands (i.e., it can be a "handheld"
device). A handheld device may have dimensions less than 15
cm.times.15 cm.times.15 cm, or less than 15 cm.times.15 cm.times.10
cm, or less than 12 cm.times.12 cm.times.6 cm. In other
embodiments, the test device 1000 can be a self-contained,
single-use device. In some embodiments, the test device 1000 can be
configured with lock-outs or other mechanisms to prevent re-use or
attempts to re-use the device.
[0117] Further, in some embodiments, the device 1000 can be a
CLIA-waived device and/or can operate in accordance with methods
that are CLIA waived. Similarly stated, in some embodiments, the
device 1000 (and any of the other devices shown and described
herein) is configured to be operated in a sufficiently simple
manner, and can produce results with sufficient accuracy to pose a
limited likelihood of misuse and/or to pose a limited risk of harm
if used improperly. In some embodiments, the device 1000 (and any
of the other devices shown and described herein), can be operated
by a user with minimal (or no) scientific training, in accordance
with methods that require little judgment of the user, and/or in
which certain operational steps are easily and/or automatically
controlled. In some embodiments, the molecular diagnostic test
device 1000 can be configured for long term storage in a manner
that poses a limited likelihood of misuse (spoilage of the
reagent(s), expiration of the reagents(s), leakage of the
reagent(s), or the like). In some embodiments, the molecular
diagnostic test device 1000 is configured to be stored for up to
about 36 months, up to about 32 months, up to about 26 months, up
to about 24 months, up to about 20 months, up to about 18 months,
or any values there between.
[0118] The test device 1000 is configured to manipulate a
biological sample S1 to produce one or more output signals
associated with a target cell. Specifically, the device 1000
includes a sample preparation module 1200, an inactivation module
1300 (also referred to as a lysing module), a fluidic drive (or
fluid transfer) module 1400, a mixing chamber 1500, an
amplification module, a detection module and a power and control
module (not shown). The test device and certain components therein
can be similar to any of the molecular test devices shown and
described herein or in International Patent Publication No.
WO2016/109691, entitled "Devices and Methods for Molecular
Diagnostic Testing," which is incorporated herein by reference in
its entirety. Accordingly, a detailed description of certain
modules (e.g., the fluidic drive module 1400) is not provided
herein. A description of each of the modules is provided below.
[0119] FIG. 11 shows a perspective exploded view of the molecular
diagnostic test device 1000. The diagnostic test device 1000
includes a housing (including a top portion 1010 and a bottom
portion 1030), within which the modules described herein are
contained. Similarly stated, the housing (including the top portion
1010 and/or the bottom portion 1030) surround and/or enclose the
modules. As shown, the top housing 1010 defines a detection opening
1011 that is aligned with the detection module 1800 such that the
signal produced by and/or on each detection surface of the
detection module 1800 is visible through the detection opening
1011. In some embodiments, the top housing 1010 and/or the portion
of the top housing 1010 surrounding the detection opening 1011 is
opaque (or semi-opaque), thereby "framing" or accentuating the
detection openings. In some embodiments, for example, the top
housing 1010 can include markings (e.g., thick lines, colors or the
like) to highlight the detection opening 1011. For example, in some
embodiments, the top housing 1010 can include indicia identifying
the detection opening to a specific disease (e.g., Chlamydia
trachomatis (CT), Neisseria gonorrhea (NG) and Trichomonas
vaginalis (TV)) or control. In other embodiments, the top housing
1010 can include a series of color spots having a range of colors
associated with a range of colors that is likely produced by the
signals produced during the test. In this manner, the housing
design can contribute to reducing the amount of user judgment
required to accurately read the test.
[0120] Referring to FIG. 11, the sample preparation module 1200
includes a sample input module 1170, a wash module 1210, an elution
module 1260, a filter assembly 1230, and various fluidic conduits
(e.g., tubes, lines, valves, etc.) connecting the various
components. The device 1000 also includes the lysing module 1300
(see e.g., the lysing module 2300 shown in FIGS. 13-16), which,
together with the sample preparation module 1200, performs the
nucleic acid extraction according to any of the methods described
herein. Thus, although the sample preparation module 1200 and the
inactivation module 1300 are described as two separate modules, in
other embodiments, the structure and function of the sample
preparation module 1200 can be included within or performed by the
inactivation module 1300 and vice-versa. Similarly stated, any of
the sample preparation modules, inactivation modules and/or lysing
modules described herein can include any of the structure and/or
perform any of the functions of the other modules to perform any of
the methods of sample preparation or nucleic acid extraction
described herein. By eliminating the need for external sample
preparation and a cumbersome instrument, the device 1000 is
suitable for use within a point-of-care setting (e.g., doctor's
office, pharmacy or the like) or at the user's home, and can
receive any suitable biological sample S1. The biological sample S1
(and any of the input samples described herein) can be, for
example, blood, urine, male urethral specimens, vaginal specimens,
cervical swab specimens, and/or nasal swab specimens gathered using
a commercially available sample collection kit.
[0121] The sample input module 1170 is disposed within the housing
1010, and is configured receive a biological sample S1 containing a
biological entity. The biological sample S1 can be any of the
sample types described herein, and the biological entity can be any
of the entities described herein. The sample input module 1170
defines a sample volume 1174 that can be selectively covered by the
cap 1152. The cap 1152 can include seals or other locking members
such that it can be securely fastened to the lower housing 1030 (or
other portions of the device 1000) and/or can be closed during
shipping, after delivery of a sample thereto, or the like. In some
embodiments, the input port cap 1152 can include an irreversible
lock to prevent reuse of the device 1000 and/or the addition of
supplemental sample fluids. In this manner, the device 1000 can be
suitably used by untrained individuals.
[0122] The wash module 1210 includes a housing that defines a wash
volume containing any suitable wash composition. For example, in
some embodiments, the wash module 1210 can include a gaseous first
wash composition (e.g., nitrogen, air, or another inert gas) and a
liquid second wash composition. In this manner, the wash operation
can include an "air purge" of the filter assembly 1230.
Specifically, when the sample input module 1170 and/or the wash
module 1210 is actuated, a serial flow of the first wash
composition (gas) followed by the second wash composition (liquid).
By first including a gas (or air) wash (i.e., the first wash
composition), the amount of liquid constituents from the input
sample conveyed to the filter assembly 1230 (indicated by the flow
S2 in FIG. 10) can be reduced. Said another way, after delivery of
the input sample, the filter assembly 1230 will retain the desired
sample cells (or organisms) and some amount of residual liquid. By
forcing the first, gaseous wash composition through the filter
(i.e., an "air wash"), the amount of residual liquid can be
minimized. This arrangement can reduce the amount of liquid wash
(e.g., the second wash composition) needed to sufficiently prepare
the sample particles. Reducing the liquid volume contributes to the
reduction size of the device 1000, and also reduces the likelihood
of potentially harmful shearing stress when the liquid wash is
flowed through the filter assembly 1230.
[0123] The sample input module 1170 (and any of the sample input
modules described herein) and the wash module 1210 (and any of the
wash modules described herein) can be actuated by any suitable
mechanism to convey the biological sample S1 towards the filter
assembly 1230 and/or the lysing module 1300 to enable the nucleic
acid extraction methods described herein. For example, in the
embodiment shown, the sample input module 1170 and the wash module
1210 are actuated by the sample actuator (or button) 1050. The
sample actuator 1050 is movably coupled to the housing, and is
aligned with and can move a piston or plunger (not shown) within
the sample volume 1174 when the sample input module 1170 is
actuated. Thus, the sample actuator 1050 is a non-electronic
actuator that is manually depressed by a user to actuate the sample
input module 1170. In other embodiments, however, the sample
actuator 1050 can be an electronic actuator. In some embodiments,
the sample actuator 1050 can include a lock tab (not shown) that is
fixedly received within the notch or opening of the housing 1010 to
fix the sample actuator 1050 in its second or "actuated" position,
as described above. In this manner, the device 1000 cannot be
reused after the initial actuation.
[0124] When actuated, the sample within the sample volume 1174 is
conveyed along with the wash solution(s) from the wash module 1210
towards the filter assembly 1230. The flow of the biological sample
S1 towards the filter assembly 1230 is shown by the arrow S2 in
FIG. 10. The filter assembly 1230 is configured to filter and
prepare the biological sample S1 (via the sample input operation
and the sample wash operation), and to allow a back-flow elution
operation to deliver captured particles from the filter membrane
and deliver the eluted volume to lysing module 1300. The filter
assembly 1230 can be toggled between two configurations to allow
the flow of the biological sample S1 and wash solution in a first
direction (towards the waste reservoir 1205), followed by a
backflush of the elution reagent and the captured organisms (or
cells) in a second direction (as indicated by the arrow S3 towards
the lysing/inactivation module 1300). The toggling mechanism can be
any suitable mechanism, such as those shown and described in
International Patent Publication No. WO2016/109691, entitled
"Devices and Methods for Molecular Diagnostic Testing," which is
incorporated herein by reference in its entirety.
[0125] The filter assembly 1230 can include any suitable filter
membrane that captures the target organism/entity while allowing
the bulk of the liquid within the biological sample S1, the first
wash composition, and the second wash composition to flow
therethrough and into the waste tank 1205. The filter membrane 1254
(and any of the filter membranes described herein) can be any
suitable membrane and or combination of membranes as described
herein. For example, in some embodiments, the filter membrane 1254
is a woven nylon filter membrane with a pore size of about 1 .mu.m
(e.g., 0.8 .mu.m, 1.0 .mu.m, 1.2 .mu.m) enclosed between various
plates of the filter assembly 1230 such that there is minimal dead
volume.
[0126] The elution module (or assembly) 1260 of the sample
preparation module 1200 is contained within the housing, and
defines an elution volume within which an elution composition is
stored. The elution composition can be any of the elution
compositions described herein. In some embodiments, the elution
composition can include proteinase K, which allows for the release
of any bound cells and/or nucleic acid molecules (e.g., DNA) from
the filter membrane. The output from the elution module 1260 can be
selectively placed in fluid communication with the filter assembly
1230, when the filter assembly is toggled into its second (or
backflow) configuration. Thus, the elution module 1230 can include
any suitable flow control devices, such as check valves, duck-bill
valves, or the like to prevent flow back towards and/or into the
elution volume.
[0127] The elution module 1210 is actuated by the elution actuator
(or button) 1070 (see FIG. 11). The reagent actuator 1070 is
movably coupled to the lower housing 1030, and can exert force on a
piston or other portion of the elution module 1210 to convey the
elution composition back through the filter and towards the lysing
module 1300, as shown by the arrow S3. In some embodiments, the
elution actuator 1070 further includes a lock tab or other
structure that is fixedly received within the notch or opening of
the housing to fix the elution actuator 1070 in its second or
"actuated" position. In this manner, the device 1000 cannot be
reused after the actuation of the elution actuator.
[0128] In use, the filter assembly 1230 recovers the target
organisms with a certain efficiency, from a given starting volume.
The wash operation then removes undesired material, without
removing the target organisms (which stay present on the filter
membrane). The elution operation then removes the target organism
from the filter membrane, diluting the total amount of captured
organisms in the volume of the elution solution, thus comprising
the eluent. By modifying the total output volume of eluent, a
higher or lower concentration of both target organism and any
potential inhibiting matter can be achieved. In some embodiments, a
further dilution can be achieved, if desired, by mixing the eluent
solution with another reagent after the initial sample preparation.
Given a known volume of eluent, and a known volume of diluent, a
correct dilution factor can be achieved, through to maintain the
reliability of the system very high dilution factors are
avoided.
[0129] As shown by the arrow S3 in FIG. 10, the elution solution
and the captured cells and/or organisms are conveyed during the
elution operation back through the filter assembly 1230, and to the
inactivation module (or lysing module) 1300. In some examples, such
as that shown by arrow S3 in FIG. 10, the elution step may involve
the nucleic acids, cells, or biological entities passing through
the filter. In other examples the elution step may involve washing
the nucleic acids, cells, or biological entities off the filter,
such that they remain on the same side of the filter without
passing through it. The inactivation module 1300 is configured to
be fluidically coupled to and receive the eluted sample S3 from the
sample preparation module 1200. In some embodiments, the
inactivation module 1300 is configured for lysis of the received
input fluid. In some embodiments, the inactivation module 1300 is
configured for de-activating the enzymes present in input fluid
after lysis occurs. In some embodiments, the inactivation module
1300 is configured for preventing cross-contamination between the
output fluid and the input fluid. The inactivation module 1300 can
include any of the inactivation (or lysing) modules as described
herein, including the lysing module 3300 and the lysing module 4300
described herein.
[0130] In some embodiments, the sample is transferred from the
inactivation module to a reverse transcription module 1900. In some
embodiments, the reverse transcription module is configured to
incubate the sample at a temperature suitable for a reverse
transcription enzyme, and subsequently incubate the sample at a
temperature high enough to deactivate the reverse transcriptase
enzyme. The reverse transcription module 1900 may include any of
the reverse transcription modules described herein.
[0131] In some embodiments the reverse transcription module 1900 is
omitted from the device and a reverse transcriptase enzyme is
present in the amplification module or the mixing module. In this
embodiment the reverse transcriptase enzyme is chosen to be one
which is active under the conditions required for the amplification
reaction. Alternatively the DNA polymerase enzyme may be chosen for
activity under the conditions required by the reverse transcriptase
enzyme. The amplification module is capable of heating the solution
to the temperatures required for reverse transcription and
inactivation of the reverse transcriptase enzyme, as well as the
temperatures required by the DNA polymerase enzyme.
[0132] The mixing module (also referred to as simply the mixing
chamber) 1500 mixes the output of inactivation module 1300 with the
reagents to conduct a successful amplification reaction. Similarly
stated, the mixing module 1500 is configured to reconstitute the
reagent in a predetermined input volume, while ensuring even local
concentrations of reagents in the entirety of the volume. In some
embodiments, the mixing chamber module 1500 is configured to
produce and/or convey a sufficient volume of liquid for the
amplification module 1600 to provide sufficient volume output to
the detection module 1800. The mixing module 1500 can be any
suitable mixing module, such as those shown and described in
International Patent Publication No. WO2016/109691, entitled
"Devices and Methods for Molecular Diagnostic Testing," which is
incorporated herein by reference in its entirety.
[0133] The fluidic drive (or transfer) module 1400 can be a pump or
series of pumps configured to produce a pressure differential
and/or flow of the solutions within the diagnostic test device
1000. Similarly stated, the fluid transfer module 1400 is
configured to generate fluid pressure, fluid flow and/or otherwise
convey the biological sample S1, and the reagents through the
various modules of the device 1000. The fluid transfer module 1400
is configured to contact and/or receive the sample flow therein.
Thus, in some embodiments, the device 1000 is specifically
configured for a single-use to eliminate the likelihood that
contamination of the fluid transfer module 1400 and/or the sample
preparation module 1200 will become contaminated from previous
runs, thereby negatively impacting the accuracy of the results. The
fluid transfer module 1500 can be any suitable fluid transfer
module, such as those shown and described in International Patent
Publication No. WO2016/109691, entitled "Devices and Methods for
Molecular Diagnostic Testing," which is incorporated herein by
reference in its entirety.
[0134] After being mixed within the mixing module 1500, the
prepared sample is then conveyed to the amplification module 1600
(as shown by the arrow CC in FIG. 10). The amplification module
1600 includes a flow member 1610 and a heater 1630. The flow member
1610 can be any suitable flow member that defines a volume or a
series of volumes within which the that prepared solution S3 can
flow and/or be maintained to amplify the target nucleic acid
molecules within the solution S3. The heater 1630 can be any
suitable heater or group of heaters coupled to the flow member 1610
that can heat the prepared solution within the flow member 1610 to
perform any of the amplification operations as described herein.
For example, in some embodiments, the amplification module 1600 (or
any of the amplification modules described herein) can be similar
to the amplification modules shown and described in U.S. patent
application Ser. No. 15/494,145, entitled "Printed Circuit Board
Heater for an Amplification Module," which is incorporated herein
by reference in its entirety. In other embodiments, the
amplification module 1600 (or any of the amplification modules
described herein) can be similar to the amplification modules shown
and described in International Patent Publication No.
WO2016/109691, entitled "Devices and Methods for Molecular
Diagnostic Testing," which is incorporated herein by reference in
its entirety.
[0135] In some embodiments, the flow member 1610 defines a single
volume within which the prepared solution is maintained and heated
to amplify the nucleic acid molecules within the prepared solution.
In other embodiments, the flow member 1610 can define a
"switchback" or serpentine flow path through which the prepared
solution flows. Similarly stated, the flow member 1610 defines a
flow path that is curved such that the flow path intersects the
heater 1630 at multiple locations. In this manner, the
amplification module 1600 can perform a "flow through"
amplification reaction where the prepared solution flows through
multiple different temperature regions.
[0136] The flow member 1610 (and any of the flow members described
herein) can be constructed from any suitable material and can have
any suitable dimensions to facilitate the desired amplification
performance for the desired volume of sample. For example, in some
embodiments, the amplification module 1600 (and any of the
amplification modules described herein) can perform 1000.lamda. or
greater amplification in a time of less than 15 minutes. For
example, in some embodiments, the flow member 1610 (and any of the
flow members described herein) is constructed from at least one of
a cyclic olefin copolymer or a graphite-based material. Such
materials facilitate the desired heat transfer properties into the
flow path. Moreover, in some embodiments, the flow member 1610 (and
any of the flow members described herein) can have a thickness of
less than about 0.5 mm. In some embodiments, the flow member 1610
(and any of the flow members described herein) can have a volume
about 150 microliters or greater, and the flow can be such that at
least 10 microliters of sample is amplified. In other embodiments,
at least 20 microliters of sample are amplified by the methods and
devices described herein. In other embodiments, at least 30
microliters of sample are amplified by the methods and devices
described herein. In yet other embodiments, at least 50 microliters
of sample are amplified by the methods and devices described
herein.
[0137] The heater 1630 can be any suitable heater or collection of
heaters that can perform the functions described herein to amplify
the prepared solution. In some embodiments, the heater 1630 can
establish multiple temperature zones through which the prepared
solution flows and/or can define a desired number of amplification
cycles to ensure the desired test sensitivity (e.g., at least 30
cycles, at least 34 cycles, at least 36 cycles, at least 38 cycles,
or at least 40 cycles). The heater 1630 (and any of the heaters
described herein) can be of any suitable design. For example, in
some embodiments, the heater 1630 can be a resistance heater, a
thermoelectric device (e.g. a Peltier device), or the like. In some
embodiments, the heater 1630 can be one or more linear "strip
heaters" arranged such that the flow path crosses the heaters at
multiple different points. In other embodiments, the heater 1630
can be one or more curved heaters having a geometry that
corresponds to that of the flow member 1610 to produce multiple
different temperature zones in the flow path.
[0138] Although the amplification module 1600 is generally
described as performing a thermal cycling operation on the prepared
solution, in other embodiment, the amplification module 1600 can
perform any suitable thermal reaction to amplify nucleic acids
within the solution. In some embodiments, the amplification module
1600 (and any of the amplification modules described herein) can
perform any suitable type of isothermal amplification process,
including, for example, Loop Mediated Isothermal Amplification
(LAMP), Nucleic Acid Sequence Based Amplification (NASBA), which
can be useful to detect target RNA molecules, Strand Displacement
Amplification (SDA), Multiple Displacement Amplification (MDA),
Ramification Amplification Method (RAM), or any other type of
isothermal process
[0139] The detection methods enabled by the device 1000 include
sequential delivery of the detection reagents and other substances
within the device 1000. Further, the device 1000 is configured to
be an "off-the-shelf" product for use in a point-of-care location
(or other decentralized location), and is thus configured for
long-term storage. Accordingly, the reagent storage module 1700 is
configured for simple, non-empirical steps for the user to remove
the reagents from their long-term storage containers, and for
removing all the reagents from their storage containers using a
single user action. In some embodiments, the reagent storage module
1700 and the rotary selection valve 1340 are configured for
allowing the reagents to be used in the detection module 1800, one
at a time, without user intervention.
[0140] Specifically, the device 1000 is configured such that the
last step of the initial user operation (i.e., the depressing of
the reagent actuator 1080) results in dispensing the stored
reagents. This action crushes and/or opens the sealed reagent
containers present in the assembly and relocates the liquid for
delivery. The rotary venting selector valve 1340 allows the reagent
module 1700 to be vented for this step, and thus allows for opening
of the reagent containers, but closes the vents to the tanks once
this process is concluded. Thus, the reagents remain in the reagent
module 1700 until needed in the detection module 1800. When a
desired reagent is needed, the rotary valve 1340 opens the
appropriate vent path to the reagent module 1700, and the fluidic
drive module 1400 applies vacuum to the output port of the reagent
module 1700 (via the detection module 1800), thus conveying the
reagents from the reagent module 1700. The reagent module 1700 and
the valve 1340 can be similar to the reagent modules and valves
shown and described in International Patent Publication No.
WO2016/109691, entitled "Devices and Methods for Molecular
Diagnostic Testing," which is incorporated herein by reference in
its entirety.
[0141] The detection module 1800 is configured to receive output
from the amplification module 1600 and reagents from the reagent
module 1700 to produce a colorimetric change to indicate presence
or absence of target organism in the initial input sample. The
detection module 1800 also produces a colorimetric signal to
indicate the general correct operation of the test (positive
control and negative control). In some embodiments, color change
induced by the reaction is easy to read and binary, with no
requirement to interpret shade or hue. The detection module 1800
can be similar to the detection modules shown and described in
International Patent Publication No. WO2016/109691, entitled
"Devices and Methods for Molecular Diagnostic Testing," which is
incorporated herein by reference in its entirety.
[0142] In one aspect, a device is provided comprising: (a) an input
port, configured to receive the biological sample comprising one or
more biological cells or biological entities; (b) a filter assembly
comprising a filter configured to capture the one or more
biological cells or biological entities, wherein the input port is
configured to relay the biological sample to the filter assembly;
(c) one or more reservoirs comprising a wash solution, a lysis
solution, or both, operably coupled to the filter assembly; (d) a
waste chamber, operably coupled to the filter assembly and
configured to receive waste from the filter assembly; and (e) an
elution chamber, operably coupled to the filter assembly and
configured to receive an eluent from the filter assembly.
[0143] For example, FIG. 12 depicts an example of a sample
preparation device (or module) 2200 that may be used to perform the
methods provided herein. The sample preparation module 2200 can be
included in any of the molecular diagnostic test devices described
herein, including the device 1000 described above. It should be
understood that the invention is not limited to a particular
arrangement or configuration of the sample preparation device, and
any suitable arrangement or configuration may be used. In some
cases, the sample preparation device 2200 comprises an input port
2170. The input port is configured to receive a sample (e.g.,
biological sample). For example, the input port 2170 may be
configured to receive about 50 .mu.L to about 20 mL of a liquid
sample. The input port 2170 may comprise a reservoir or chamber for
holding or storing the sample. The input port 2170 may comprise a
cap or lid (similar to the lid 1152 described above) that can be
placed over the input port to contain the sample in the reservoir
or chamber. The input port 2170 may be operably coupled to a filter
assembly 2230. In use, the sample may be relayed (e.g., pushed or
flowed) to the filter assembly 2230 in any manner as described
herein. The filter assembly 2230 may contain one or more filter
membranes for capturing biological cells or entities on the filter.
In some instances, the filter assembly 2230 (or any of the filter
assemblies described herein) contains at least two filter
membranes, one with a larger pore size and one with a smaller pore
size. The two filter membranes may be arranged such that the sample
first passes through the membrane with the larger pore size and
then the membrane with the smaller pore size. The filter membrane
may be of any suitable material as described herein, non-limiting
examples including nylon, cellulose, polyethersulfone (PES),
polyvinylidene difluoride (PVDF), polycarbonate, borosilicate glass
fiber and the like. In some examples, the filter membrane is nylon.
In some cases, the filter membrane has an average pore size of
about 0.2 .mu.m to about 20 .mu.m. For example, the filter membrane
may have an average pore size of about 0.2 .mu.m, about 0.5 .mu.m,
about 1 .mu.m, about 2 .mu.m, about 3 .mu.m, about 4 .mu.m, about 5
.mu.m, about 6 .mu.m, about 7 .mu.m, about 8 .mu.m, about 9 .mu.m,
about 10 .mu.m, about 11 .mu.m, about 12 .mu.m, about 13 .mu.m,
about 14 .mu.m, about 15 .mu.m, about 16 .mu.m, about 17 .mu.m,
about 18 .mu.m, about 19 .mu.m, about 20 .mu.m, or greater than 20
.mu.m. In some examples, the surface of the filter membrane may be
chemically treated or coated in such a way as to improve the
binding of a biological cell or entity to the membrane. The
biological cells or entities may be captured on the membrane while
the majority of the liquid ("flow-through") is flowed through the
filter membrane. In some cases, the flow-through is substantially
devoid of biological cells or entities. In some cases, the
flow-through is disposed of by relaying the flow-through to one or
more waste chambers operably coupled to the filter assembly. In
other cases, the flow-through is relayed to a collection chamber
for further downstream processing.
[0144] In some aspects, the sample preparation device 2200 further
comprises one or more chambers 2210 or reservoirs for housing a
wash solution. The one or more chambers or reservoirs (also
referred to as wash modules) housing the wash solution may be
operably coupled to the filter assembly such that actuation of the
wash chamber or reservoir 2210 relays the wash solution to the
filter assembly 2230. In some cases, the wash solution is provided
as a lyophilized pellet or bead that sits within the chamber or
reservoir. The lyophilized pellet or bead can be reconstituted in
one or more solutions. The wash solution may be flowed through the
filter assembly 2230 and the majority of the liquid can be
collected in the one or more waste chambers 2205. Non-limiting
examples of wash solutions suitable for use with the sample
preparation device have been described above.
[0145] In certain aspects, the sample preparation device further
comprises one or more chambers or reservoirs for housing a lysis
solution. The chamber or reservoir housing the lysis solution may
be operably coupled to the filter assembly such that actuation of
the chamber or reservoir relays the lysis solution to the filter
assembly. In some cases, the lysis solution may be flowed through
the filter assembly. The lysis solution may cause the lysis or
disruption of the biological cells or entities on the filter
membrane. In some cases, the reagents of the lysis solution are
provided as a lyophilized pellet or bead that sits within the
chamber or reservoir (e.g., within a lysing module, similar to the
lysing modules 1300, 3300 and 4300 described herein). The
lyophilized pellet or bead can be reconstituted in one or more
solutions. In some cases, the lysis enzyme is stored separately as
a lyophilized bead or pellet within the device. In some cases, the
lyophilized lysis enzyme may be reconstituted in the lysis buffer
prior to addition to the cells. In other cases, the cells are
eluted from the filter membrane and relayed into the elution
chamber 2260 which contains the lyophilized lysis enzyme, thereby
reconstituting the enzyme. In cases where a lysis enzyme is used,
the enzyme is stable in the device at ambient temperatures for long
periods of time. For example, the enzyme may be stable in the
device at ambient temperature for at least one day, at least two
days, at least three days, at least four days, at least five days,
at least six days, at least one week, at least two weeks, at least
three weeks, at least four weeks, at least a month, at least two
months, at least three months, at least four months, at least five
months, at least six months, at least seven months, at least eight
months, at least nine months, at least ten months, at least eleven
months, at least one year, at least two years, at least three
years, at least four years, at least five years, at least six
years, at least seven years, at least eight years, at least nine
years, at least ten years or longer. The lysis solution containing
the lysed cells ("eluent") may be collected in an elution chamber.
In some cases, the lysis solution may be back-flowed through the
filter assembly. In this instance, the biological cells or entities
on the filter membrane may be pushed or washed from the membrane
and collected in an elution chamber with the lysis solution. The
cells or entities (or lysed or otherwise disrupted cells or
entities) diluted in the lysis solution may be referred to as the
"eluent."
[0146] In some aspects, the sample preparation device 2200 may
further comprise one or more heating modules (not shown). The one
or more heating modules may be operably coupled to the elution
chamber 2260. The one or more heating modules may heat the elution
chamber to a temperature sufficient for lysis of the biological
cells or entities to occur. In some cases, the lysis solution
comprises one or more enzymes (e.g., Proteinase K). In some cases,
the one or more heating modules heats the elution chamber to a
temperature sufficient for optimal performance of the lysis enzyme.
In some examples, the heating module heats the elution chamber (and
the fluid contained therein) to a temperature of about 4.degree.
C., about 10.degree. C., about 15.degree. C., about 20.degree. C.,
about 25.degree. C., about 30.degree. C., about 40.degree. C.,
about 45.degree. C., about 50.degree. C., about 55.degree. C.,
about 60.degree. C., about 65.degree. C., about 70.degree. C.,
about 75.degree. C. or greater than 75.degree. C.
[0147] In some aspects, the sample preparation device 2200 and/or
any of the molecular diagnostic devices described herein further
comprises an inactivation chamber (also referred to as an
inactivation module or a lysing module). The inactivation chamber
may be operably coupled to the elution chamber. The eluent may be
relayed from the elution chamber to the inactivation chamber. In
some instances, the elution chamber and the inactivation chamber
are the same chamber and are coupled to a heating element that can
heat the chamber to an optimal lysis temperature, and can further
heat the chamber to an optimal inactivation temperature (e.g., from
about 56.degree. C. to about 95.degree. C.).
[0148] For example, a non-limiting example of an inactivation
chamber 3300 is depicted in FIGS. 13-16. In this example, the
inactivation chamber comprises a chamber body 3310, a bottom lid
3318, and a heater 3330. As depicted in FIG. 12, the chamber body
3310 may defines an input port 3312, a holding tank (or first
volume) 3311, a permanent vent 3314, an inactivation segment (or
second volume) 3321, and an output port 3313. The input port 3312
may be configured to receive the eluent from the elution chamber
and/or directly from a filter assembly (e.g., the filter assembly
1230). In other embodiments, as described herein, the input port
3312 can be fluidically coupled to a sample input module without
the biological input being conveyed through a filter. The eluent
may flow into the inactivation chamber (or lysing module 3300) and
be collected in the holding tank 3311. The holding tank may have a
capacity of about 1 .mu.L to about 100 mL, about 100 .mu.L to about
10 mL, about 300 .mu.L to 1 mL, or about 300 .mu.L to about 650
.mu.L. The holding tank may be used to lyse the sample. For
example, in some embodiments, the eluent containing the target
organisms can be heated by the heater 3330 to maintain the eluent
at or above a target lysing temperature. Similarly stated, in some
embodiments, the heater 3330 can be coupled to the chamber body
3310 and/or the bottom lid 3318 such that the heater 3330 can
convey thermal energy into the lysing module 3300 to produce a
lysing temperature zone within the holding tank (or first volume)
3311. The lysing temperature zone can maintain the eluent at any of
the temperatures and for any of the time periods described
herein.
[0149] The vent 3314 may be a hole which allows air to flow into or
out of the lysing module 3300 (including the first volume 3311 and
the second volume 3321) as sample is brought in or out. The vent
3314 can also relieve pressure within either of the first volume
3311 or the second volume 3321 when the eluent is heated. Although
described as being a permanent vent (i.e., a vent having a fixed
opening), in some embodiments, the lysing module 3300 (or any of
the lysing modules described herein) can have an active vent. For
example, in some embodiments, the lysing module 3300 (or any of the
lysing modules described herein) can include a valve that controls
the venting of pressure and/or air from within the lysing module
3300.
[0150] The eluent may flow from the holding tank 3311 through the
inactivation segment of the lysing module 3300. More specifically,
the holding tank 3311 is in fluid communication with the
inactivation segment 3321 such that when a pressure gradient is
applied across the input port 3312 and the output port 3313, the
eluent can flow from the holding tank 3311 (first volume) through
the inactivation segment 3321 (second volume). The pressure
gradient can be applied by any suitable mechanism, such as for
example, a pump (e.g., the fluidic drive module 1400). The
inactivation segment 3321 may be a small, shallow channel that
allows efficient and rapid heating of the eluent as it leaves the
holding tank. In a non-limiting example, the inactivation segment
3321 is configured in a serpentine pattern. The serpentine pattern
may allow for rapid inactivation of the lysis enzymes in the
eluent. The eluent, after being flowed through the inactivation
segment, may be flowed into the output port 3313 to be collected.
The volume of liquid passed through the heated channel could be
from about 1 .mu.L to about 100 mL, about 10 .mu.L to about 10 mL,
about 100 to about 5 mL, or about 250 .mu.L to about 750 .mu.L.
[0151] As described above, the inactivation module 3300 may be in
contact with a heating element 3330, which can be, for example, a
printed circuit board (PCB) heater. The heating element 3330 may
function to heat the eluent as it flows through the inactivation
segment at a high temperature sufficient to inactivate the one or
more lysis enzymes contained within the eluent. For example, the
heating element may heat the eluent to about 57.degree. C., about
58.degree. C., about 59.degree. C., about 60.degree. C., about
61.degree. C., about 62.degree. C., about 63.degree. C., about
64.degree. C., about 65.degree. C., about 66.degree. C., about
67.degree. C., about 68.degree. C., about 69.degree. C., about
70.degree. C., about 71.degree. C., about 72.degree. C., about
73.degree. C., about 74.degree. C., about 75.degree. C., about
76.degree. C., about 77.degree. C., about 78.degree. C., about
79.degree. C., about 80.degree. C., about 81.degree. C., about
82.degree. C., about 83.degree. C., about 84.degree. C., about
85.degree. C., about 86.degree. C., about 87.degree. C., about
88.degree. C., about 89.degree. C., about 90.degree. C., about
91.degree. C., about 92.degree. C., about 93.degree. C., about
94.degree. C., about 95.degree. C., about 96.degree. C., about
97.degree. C., about 98.degree. C., about 99.degree. C., about
100.degree. C. or greater than 100.degree. C. By heating the liquid
eluent to a high temperature, the lysis enzymes as well as any
other enzymes present can be deactivated. In some embodiments, the
sample can be heated to about 95 C for about 3 minutes. In some
embodiments, the serpentine path 3321 may be preceded by a check
valve (not shown) to maintain a back pressure such that fluid does
not enter the serpentine path 3321 before the desired temperature
has been achieved. The serpentine area may be preheated to the
desired temperature (50.degree. C. to 99.degree. C. or more) before
fluid is drawn through the serpentine channel. If fluid were to
flow into the serpentine channel prematurely without controlled
flow, large bubbles may form in the channel as the heater warms up
which could result in portions of the fluid to pass through the
channel without receiving the proper temperature treatment.
In some embodiments there may be a one-way check valve that allows
flow between the inactivation chamber and the mixing chamber (and
prevents reverse flow). However, before flow can occur a certain
amount of "cracking pressure" must be achieved. If the holding tank
of the inactivation chamber is well vented from a vent port, the
liquid that is placed into the holding tank will not flow into the
serpentine channel due to the cracking pressure of the check valve
at the exit of the serpentine channel. The cracking pressure may be
from 0.05 to 50 psi. In some examples, the check valves used may
have a cracking pressure of approximately 0.5 psi.
[0152] As described, the solution within the second volume 3321 is
rapidly heated to temperatures of up to about 100 degrees Celsius.
The lysing module 3300 and/or the formulation of the input solution
(e.g., the eluent), however, can collectively reduce the likelihood
that the liquid portion of the input solution will boil during the
lysing/inactivation operations. Such boiling can produce
undesirable bubbles and/or air pockets and can reduce the
repeatability of the lysing and/or inactivation operations.
Moreover, to facilitate use of the device at a variety of different
altitudes, the lysing module 3300 and/or the formulation of the
input solution can collectively reduce the likelihood that the
liquid portion of the input solution will boil at a temperature of
99 degrees Celsius or higher, 98 degrees Celsius or higher, 96
degrees Celsius or higher, 94 degrees Celsius or higher, 92 degrees
Celsius or higher, 90 degrees Celsius or higher, or 88 degrees
Celsius or higher. For example, in some embodiments, the input
solution can include salts and/or sugars to raise the boiling
temperature of the input solution. In other embodiments, the lysing
module 3300 can include one or more vent openings into either the
first volume 3311 or the second volume 3321 or both (to limit
pressure build-up during heating).
[0153] After the lysing and inactivation operations, the output
from the lysing module 3300 can be conveyed into an (e.g., the
amplification module 1600 or any other amplification modules
described herein). Similarly stated, the output from the lysing
module 3300, which contains the extracted nucleic acid molecules,
can be conveyed to an amplification module. The amplification
module can then perform a thermal reaction (e.g., an amplification
reaction) on the prepared solution containing target nucleic acid
mixed with required reagents. In some embodiments, the
amplification module is configured to conduct rapid amplification
of an input target. In some embodiments, the amplification module
is configured to generate an output copy number that reaches or
exceeds the threshold of the sensitivity of an associated detection
module (e.g., the detection module 1800).
[0154] FIGS. 17-22 show various views of a lysing module 4300 (also
referred to as an inactivation module), according to an embodiment.
The lysing module 4300 includes a chamber body 4310, a bottom lid
4318, a heater 4330, and an electrode assembly. The chamber body
4310 and the bottom lid 4318 can be referred to as a flow member.
Although the flow member is shown as being constructed from two
pieces (the body 4310 and the bottom lid 4318) that are coupled
together, in other embodiments, the flow member can be
monolithically constructed. The chamber body 4310 and the bottom
lid 4318 define an input port 4312, a first (or holding) volume
4311, a vent 4314, a second (or inactivation) volume 4321, and an
output port 4313. The input port 4312 can receive the eluent from
the elution chamber and/or directly from a filter assembly (e.g.,
the filter assembly 1230). In other embodiments, as described
herein, the input port 4312 can be fluidically coupled to a sample
input module without the biological input being conveyed through a
filter. In use, the eluent can flow into the lysing module 4300 and
be collected in the holding volume 4311. The sample can be lysed
within the holding volume 4311. For example, in some embodiments,
the eluent containing the target organisms can be heated by the
heater 4330 to maintain the eluent at or above a target lysing
temperature. Similarly stated, in some embodiments, the heater 4330
can be coupled to the chamber body 4310 and/or the bottom lid 4318
such that the heater 4330 can convey thermal energy into the lysing
module 4300 to produce a lysing temperature zone within the holding
volume 4311. The lysing temperature zone can maintain the eluent at
any of the temperatures and for any of the time periods described
herein.
[0155] The vent opening 4314 is in fluid communication with the
first volume 4311, and thus allows air to flow into or out of the
lysing module 4300 (including the first volume 4311 and the second
volume 4321) as sample is conveyed into and/or out of the lysing
module 4300. The vent 4314 can also relieve pressure within either
of the first volume 4311 or the second volume 4321 when the eluent
is heated. Although shown as being a permanent vent (i.e., a vent
having a fixed opening), in some embodiments, the lysing module
4300 (or any of the lysing modules described herein) can have an
active vent. For example, in some embodiments, the lysing module
4300 (or any of the lysing modules described herein) can include a
valve that controls the venting of pressure and/or air from within
the lysing module 4300.
[0156] The first volume 4311 is in fluid communication with the
second volume 4322. In this manner, the eluent can flow from the
first (or holding) volume 4311 through the second (or inactivation)
volume 4321 of the lysing module 4300. More specifically, when a
pressure gradient is applied across the input port 4312 and the
output port 4313, the eluent can flow from the holding volume 4311
(first volume) through the second volume 4322. The pressure
gradient can be applied by any suitable mechanism, such as for
example, a pump (e.g., the fluidic drive module 1400). As shown,
the second volume 4321 is a serpentine channel that provides a high
surface area to volume ratio. This arrangement allows for rapid
inactivation of the lysis enzymes in the eluent. The eluent, after
being flowed through the inactivation segment, may be flowed into
the output port 4313 to be collected and/or conveyed to an
amplification module (not shown).
[0157] As described above, the flow member is in contact with a
heating element 4330, which can be, for example, a printed circuit
board (PCB) heater. The heating element 4330 may function to heat
the eluent as it flows through the second volume 4311 at a high
temperature sufficient to inactivate the one or more lysis enzymes
contained within the eluent. For example, the heating element may
heat the eluent to about 57.degree. C., about 58.degree. C., about
59.degree. C., about 60.degree. C., about 61.degree. C., about
62.degree. C., about 63.degree. C., about 64.degree. C., about
65.degree. C., about 66.degree. C., about 67.degree. C., about
68.degree. C., about 69.degree. C., about 70.degree. C., about
71.degree. C., about 72.degree. C., about 73.degree. C., about
74.degree. C., about 75.degree. C., about 76.degree. C., about
77.degree. C., about 78.degree. C., about 79.degree. C., about
80.degree. C., about 81.degree. C., about 82.degree. C., about
83.degree. C., about 84.degree. C., about 85.degree. C., about
86.degree. C., about 87.degree. C., about 88.degree. C., about
89.degree. C., about 90.degree. C., about 91.degree. C., about
92.degree. C., about 93.degree. C., about 94.degree. C., about
95.degree. C., about 96.degree. C., about 97.degree. C., about
98.degree. C., about 99.degree. C., about 100.degree. C. or greater
than 100.degree. C. By heating the liquid eluent to a high
temperature, the lysis enzymes as well as any other enzymes present
can be deactivated. In some embodiments, the sample can be heated
to about 95 C for about 4 minutes.
[0158] In some embodiments the heater on the PCB 4330 is
specifically designed to heat the serpentine portion of the lysing
module 4300 (i.e., the second volume 4321) while not heating the
holding volume 4311. Because the lid 4318 of the lysing module 4300
is thick, the heater surface may be heated well above the desired
temperature of the fluid. Since the electrodes 1971, 1972
(described in more detail below) are thermally conductive and come
into direct contact with the fluid, the fluid surrounding the
electrodes 1971, 1972 will experience the same temperature as the
heater surface, which may cause evaporation. To minimize the
heating of the holding volume 4311, a slot (not shown) may be cut
in the PCB 4330 to isolate the heater from the portion of the PCB
adjacent and/or in contact with the holding volume 4311. For
example, in some embodiments, the heater 4330 can include a series
of slots and/or openings as described in U.S. patent application
Ser. No. 15/494,145, entitled "Printed Circuit Board Heater for an
Amplification Module," which is incorporated herein by reference in
its entirety. Moreover, in some embodiments, the heating element of
the heater 4330 is located on an internal layer so the top copper
pour (not shown) can be used as a heat spreader to minimize
temperature variation along the serpentine path. The six wires
soldered to the PCB 4330 may remove heat from the surrounding area,
creating temperature gradients across the heater surface. To
minimize this effect, wires may be soldered on both sides of the
heater surface so the temperature roll off is symmetrical.
[0159] In some embodiments, the lysing module 4300 can determine
whether there is liquid in the first volume 4311 and/or the second
volume 4321. Specifically, the lysing module 4300 includes
electrical probes to determine electrical resistance of the fluid
within the first volume. In some embodiments, the molecular
diagnostic device (e.g., the device 1000) can include an electronic
controller configured to determine when the user has actuated the
elution module (e.g., by pressing an elution actuator, similar to
the button 1070 described above) by detecting the presence of
liquid in the first volume 4311. In this manner, the introduction
of liquid into the first volume 4311 can trigger the start of the
device.
[0160] Specifically, the control system and/or the lysing module
4300 includes two electrodes 4971, 4972 inside the first volume
4311. The electrodes 4971, 4972 are connected to circuitry (e.g., a
controller, not shown) that detects a resistance change between the
two electrodes 4971, 4972. Fluid may be reliably detected between
the electrodes 4971, 4972 due to the high gain of the circuit,
which may easily differentiate between an open circuit condition
(no fluid) and a non-negligible resistance across the electrodes
4971, 4972 (fluid detected). Use of a sample matrix with high salt
concentration increases the conductivity of the fluid, which may
make the fluid easily detectable even with variation across
samples.
[0161] The electrodes 4971, 4972 and the circuitry (not shown) are
designed to detect fluid without impacting the biological processes
that take place in the device. For example, the electrodes 4971,
4972 are specifically chosen so as not inhibit PCR reactions. In
some embodiments, the electrodes 4971, 4972 are gold plated.
[0162] Both DNA and cells have a net charge so they may migrate in
the presence of an electric field. Because the resistance change
between the electrodes 4971, 4972 is determined by measuring a
change in electric potential, precautions may be taken to minimize
the impact of this electromotive force. For example, once fluid is
detected voltage may be removed from the electrodes 4971, 4972 and
they may be electrically shorted together. This ensures there is no
potential difference between the electrodes 4971, 4972 and the
charged particles (DNA, cells, salts, etc.) will not bind to the
electrodes, which would prevent them from entering the
amplification module (not shown).
[0163] As described, the solution within the second volume 4321 is
rapidly heated to temperatures of up to about 100 degrees Celsius.
The lysing module 4300 and/or the formulation of the input solution
(e.g., the eluent), however, can collectively reduce the likelihood
that the liquid portion of the input solution will boil during the
lysing/inactivation operations. Such boiling can produce
undesirable bubbles and/or air pockets and can reduce the
repeatability of the lysing and/or inactivation operations.
Moreover, to facilitate use of the device at a variety of different
altitudes, the lysing module 4300 and/or the formulation of the
input solution can collectively reduce the likelihood that the
liquid portion of the input solution will boil at a temperature of
99 degrees Celsius or higher, 98 degrees Celsius or higher, 96
degrees Celsius or higher, 94 degrees Celsius or higher, 92 degrees
Celsius or higher, 90 degrees Celsius or higher, or 88 degrees
Celsius or higher. For example, in some embodiments, the input
solution can include salts and/or sugars to raise the boiling
temperature of the input solution. In other embodiments, the lysing
module 4300 can include one or more vent openings into either the
first volume 4311 or the second volume 4321 or both (to limit
pressure build-up during heating).
[0164] The reverse transcription module consists of an incubation
chamber in which a reverse transcription reaction can take place
and a means to heat the sample to a temperature sufficient to
deactivate a reverse transcriptase enzyme. The reverse
transcriptase may be present as a lyophilized pellet in the
incubation chamber of the reverse transcription module. The
lyophilized pellet is rehydrated by the sample when the sample
enters 1900, thus allowing a reverse transcription reaction to
occur. The lyophilized pellet may contain suitable salts to buffer
the sample to ensure suitable conditions for the reverse
transcriptase enzyme. In some cases the reverse transcription
enzyme may be chosen to have activity in the sample without
requiring additional buffers. The lyophilized pellet may also
contain compounds of additives to stabilize the enzyme in the
lyophilized state and preserve enzymatic activity once rehydrated.
The lyophilized pellet may contain primers for the reverse
transcriptase enzyme. The primers may be specific primers to
amplify RNA molecules of specific sequences, random primers such as
random hexamers, or primers targeted to common sequences, such as
poly T primers to amplify RNA molecules with poly-A tails.
[0165] The reverse transcription reaction may occur in the
incubation chamber of the reverse transcription module 1900. The
incubation chamber may be
[0166] After the lysing and inactivation operations, the output
from the lysing module 4300 can be conveyed into an (e.g., the
amplification module 1600 or any other amplification modules
described herein). Similarly stated, the output from the lysing
module 4300, which contains the extracted nucleic acid molecules,
can be conveyed to an amplification module. The amplification
module can then perform a thermal reaction (e.g., an amplification
reaction) on the prepared solution containing target nucleic acid
mixed with required reagents. In some embodiments, the
amplification module is configured to conduct rapid amplification
of an input target. In some embodiments, the amplification module
is configured to generate an output copy number that reaches or
exceeds the threshold of the sensitivity of an associated detection
module (e.g., the detection module 1800).
[0167] Although the device shown in FIG. 10 is described as
including a filter assembly, in some embodiments, a sample
preparation device need not include a filter or filter assembly.
For example, in some embodiments the sample input may be directly
linked to an inactivation chamber, as shown schematically in FIG.
23. Advantages of a device without a filter assembly include lower
pressures in the device, no risk of breaking a filter, fewer parts,
fewer reagents required, higher recovery of target organisms from
the clinical sample matrix and higher recovery of DNA from target
organisms. FIG. 23 and FIG. 35 shows a portion of a molecular test
device 5000 that includes a sample input module 5170 and an
inactivation (or lysing) module 5300. The portion of a molecular
test device in FIG. 35 further comprises a reverse transcription
module 5600. The device 5000 can be similar to the device 1000
described above, and can include an amplification module, a
detection module or the like. In this case, the device 5000 differs
from the device 1000 in that the sample is flowed from the input
module 5170 into the holding tank of the inactivation module 5300.
The sample may be lysed either in the holding tank 5311 or in the
inactivation segment 5321. In this case the sample may be lysed by
heating without need for a specialized lysis buffer or lysis
enzymes. Any proteases or nucleases released from the cells of the
sample will be inactivated by heating. For example, a sample may be
flowed into the holding tank and held until the inactivation
segment 5321 reaches a set temperature (for example greater than 90
C) and then flowed through the inactivation segment. In the
inactivation segment the sample is rapidly heated to 95 C causing
the cells in the sample to lyse and proteins from within the cells
to be inactivated. The sample may be reverse transcribed in the
reverse transcription chamber 5611 and the reverse transcriptase
enzyme may be inactivated in the inactivation segment 5621.
[0168] As another example of an embodiment in which the sample is
not conveyed through a filter, FIG. 24 is a schematic illustration
of a molecular diagnostic test device 6000 (also referred to as a
"test device" or "device"), according to an embodiment. The test
device 6000 includes a housing 6010, a sample input module 6170, a
lysing module 6300, and an amplification module 6600. The housing
6010 can be any structure within which the sample input module
6170, the lysing module 6300, and the amplification module 6600 are
contained. In some embodiments, the test device 6000 can have a
size, shape and/or weight such that the device can be carried,
held, used and/or manipulated in a user's hands (i.e., it can be a
"handheld" device). In other embodiments, the test device 6000 can
be a self-contained, single-use device of the types shown and
described herein (e.g., the device 1000) or in International Patent
Publication No. WO2016/109691, entitled "Devices and Methods for
Molecular Diagnostic Testing," which is incorporated herein by
reference in its entirety.
[0169] The sample input module 6170 is disposed within the housing
6010, and is configured receive a biological sample S1 containing a
biological entity. The biological sample S1 can be any of the
sample types described herein, and the biological entity can be any
of the entities described herein. The sample input module 6170
defines a sample volume 6174, and includes a piston 6180 that is
movably disposed within the sample volume 6174. In use the
biological sample S1 can be conveyed into the sample volume 6174 by
any suitable mechanism, such as, for example, via a pipette, a
dropper, or the like. In some embodiments, the biological sample S1
can be conveyed via an opening into the sample volume 6174 that can
be blocked to prevent backflow of the sample back out of the sample
input volume 6174. For example, in some embodiments, the sample
input module 6170 can include any suitable flow control devices,
such as check valves, duck-bill valves, or the like, to control the
flow of the biological sample S1 within the device 6000.
[0170] The sample input module 6170 (and any of the sample input
modules described herein) can be actuated by any suitable mechanism
to convey the biological sample S1 towards the lysing module 6300
to enable the nucleic acid extraction methods described herein. For
example, in the embodiment shown, the sample input module 6170 is
actuated by the sample actuator (or button) 6050. The sample
actuator 6050 is movably coupled to the housing 6010, and is
aligned with and can move the piston 6180 when the sample input
module 6170 is actuated. The sample actuator 6050 is a
non-electronic actuator that is manually depressed by a user to
actuate the sample input module 6170. In other embodiments,
however, the sample actuator 6050 can be an electronic actuator. In
some embodiments, the sample actuator 6050 can include a lock tab
(not shown) that is fixedly received within the notch or opening of
the housing 6010 to fix the sample actuator 6050 in its second or
"actuated" position, as described above. In this manner, the device
6000 cannot be reused after the initial actuation. When the piston
6180 is moved downward within the sample volume 6174, as shown by
the arrow AA, the sample within the sample volume 6174 is conveyed
towards the lysing module 6300. The flow of the biological sample
S1 towards the lysing module 6300 is shown by the arrow S2 in FIG.
24.
[0171] The lysing module 6300 (also referred to as the inactivation
module), which can be a portion of a sample preparation module, is
configured to process the biological sample S1 to facilitate
detection of an organism therein that is associated with a disease.
Specifically, the lysing module 6300 is configured to concentrate
and lyse cells in the biological sample S1, thereby allowing
subsequent extraction of a nucleic acid to facilitate amplification
(e.g., via the amplification module 6600) and/or detection (e.g.,
via a detection module, not shown). As shown, the processed/lysed
sample (e.g., the sample S3) is pushed and/or otherwise transferred
from the lysing module 6300 to other modules within the device 6000
(e.g., the amplification module 6600). By eliminating the need for
external sample preparation and a cumbersome instrument, the device
6000 is suitable for use within a point-of-care setting (e.g.,
doctor's office, pharmacy or the like) or at the user's home, and
can receive any suitable biological sample S1. The biological
sample S1 (and any of the input samples described herein) can be,
for example, blood, urine, male urethral specimens, vaginal
specimens, cervical swab specimens, and/or nasal swab specimens
gathered using a commercially available sample collection kit.
[0172] The lysing module includes a flow member 6310 and a heater
6330. The flow member 6310 includes an input port 6312 and an
output port 6313, and defines a first volume 6311 and a second
volume 6321. As shown, the first volume 6311 can receive an input
solution (identified as S2) containing at least the biological
sample S1 and a lysis buffer. The lysis buffer can be any of the
lysis buffers described herein. Moreover, the lysis buffer can be
mixed with the biological sample S1 to form the input solution S2
in any suitable manner or at any suitable location within the
device 6000. For example, in some embodiments, the lysis buffer can
be stored within the sample input module 6170, and can be mixed
with the biological sample S1 when the biological sample S1 is
conveyed into the volume 6174. In other embodiments, the lysis
buffer can be stored in a reagent module (not shown) and can be
mixed with the biological sample S1 when the sample input module
6170 is actuated (e.g., via the actuator 6050). In yet other
embodiments, the lysis buffer can be stored in the lysing module
6300 (e.g., the first volume 6311).
[0173] The heater 6330 is coupled to the flow member 6310 and is
configured to produce thermal energy that is conveyed into the
first volume 6311, the second volume 6321, or both the first volume
6311 and the second volume 6321 to lyse organisms within the
biological sample S1 and/or the input solution S2. In this manner,
the lysing module 6300 can release one or more nucleic acid
molecules from within the cells and/or organisms within the
biological sample S1 and/or the input solution S2. Specifically,
the heater 6330 and the flow member 6310 are collectively
configured to maintain the input solution S2 at a desired lysing
temperature for a predetermined amount of time to facilitate and/or
promote lysing of the organisms therein. For example, in some
embodiments, the first volume 6311 and/or the second volume 6321
can be maintained at a temperature between about 55 degrees Celsius
and about 600 degrees Celsius for a time period of about 25 seconds
or more. In other embodiments, the first volume 6311 and/or the
second volume 6321 can be maintained at a temperature between about
92 degrees Celsius and about 98 degrees Celsius.
[0174] In addition to lysing organisms within the input solution S2
to release nucleic acid molecules, the heater 6330 and the flow
member 6310 are configured to heat the first volume 6311, the
second volume 6321, or both the first volume 6311 and the second
volume 6321 to inactivate enzymes present within the biological
sample S1 and/or the input solution S2. Specifically, by heating
the input solution S2, the lysing module 6300 can denature certain
proteins and/or inactivate certain enzymes present within organisms
that are within the input solution S2. Such proteins and/or enzymes
can, in certain instances, limit the efficiency or effectiveness of
the desired amplification operation. Thus, rapid and efficient
inactivation can improve the repeatability and accuracy of the
amplification and/or the detection of the molecular diagnostic
device 6000. In some embodiments, for example, the heater 6330 and
the flow member 6310 can collectively produce an inactivation
temperature zone within which the input solution S2 can be heated
to within the desired temperature range and/or for the desired time
period to produce the desired inactivation. For example, in some
embodiments, the input solution S2 within the lysing module 6300
can be maintained at a temperature between about 55 degrees Celsius
and about 600 degrees Celsius for a time period of about 25 seconds
or more. In other embodiments, the input solution S2 within the
lysing module 6300 can be maintained at a temperature between about
92 degrees Celsius and about 98 degrees Celsius.
[0175] Although described as occurring in two separate heating
operations, the lysing and the inactivation can be performed by a
single heating operation. For example, in some embodiments, the
input solution S2 can be heated to the desired temperature range to
both lyse the organisms and inactivate the enzymes as the input
solution S2 flows through the first volume 6311 and/or the second
volume 6321. Said another way, in some embodiments, the lysing
module 6300 can perform "flow through" inactivation and lysing
operations. For example, in some embodiments, either of the first
volume 6311 or the second volume 6321 (or both) can define a
tortuous flow path through which the input solution S2 flows during
the lysing/inactivation operation. In this manner, the surface
area-to-volume ratio of the first volume 6311 and/or the second
volume 6321 can be high enough such that the heat transfer into the
input solution S2 occurs rapidly as it flows through the lysing
module. In some embodiments, for example, the first volume 6311
and/or the second volume 6321 can define a serpentine flow path. In
some embodiments, a ratio of the surface area of the second volume
6321 to the volume of the second volume 6321 is 20 cm.sup.-1.
[0176] In some embodiments, the flow member 6310 (and any of the
flow members described herein) can have a volume about 650
microliters or greater, and the flow can be such that at least 60
microliters of the input solution S2 is prepared for amplification
(i.e., has nucleic acids extracted therefrom). In other
embodiments, at least 20 microliters of the input solution S2 is
prepared for amplification by the methods and devices described
herein. In other embodiments, at least 30 microliters of the input
solution S2 is prepared for amplification by the methods and
devices described herein. In yet other embodiments, at least 50
microliters of the input solution S2 is prepared for amplification
by the methods and devices described herein.
[0177] As described above, in some embodiments, the input solution
S2 is rapidly heated to temperatures of up to about 100 degrees
Celsius. The lysing module 6300 and/or the formulation of the input
solution S2, however, can collectively reduce the likelihood that
the liquid portion of the input solution S2 will boil during the
lysing/inactivation operations. Such boiling can produce
undesirable bubbles and/or air pockets and can reduce the
repeatability of the lysing and/or inactivation operations.
Moreover, to facilitate use of the device at a variety of different
altitudes, the lysing module 6300 and/or the formulation of the
input solution S2 can collectively reduce the likelihood that the
liquid portion of the input solution S2 will boil at a temperature
of 99 degrees Celsius or higher, 98 degrees Celsius or higher, 96
degrees Celsius or higher, 94 degrees Celsius or higher, 92 degrees
Celsius or higher, 90 degrees Celsius or higher, or 88 degrees
Celsius or higher. For example, in some embodiments, the input
solution S2 can include salts and/or sugars to raise the boiling
temperature of the input solution S2. In other embodiments, the
lysing module 6300 can include one or more vent openings into
either the first volume 6311 or the second volume 6321 or both (to
limit pressure build-up during heating). In such embodiments, the
vent opening can be such that a limited amount of pressure is
allowed within the first volume 6311 or the second volume 6321 to
raise the boiling temperature of the input solution S2.
[0178] After the lysing and inactivation operations, the output
from the lysing module 6300 can be conveyed into the amplification
module 6600. Similarly stated, the output from the lysing module
6300, which is identified as the prepared solution S3, and which
contains the extracted nucleic acid molecules, can be conveyed to
the amplification module 6600. The amplification module 6600 can
then perform a thermal reaction (e.g., an amplification reaction)
on the prepared solution S3 containing target nucleic acid mixed
with required reagents. In some embodiments, the amplification
module 6600 is configured to conduct rapid amplification of an
input target. In some embodiments, the amplification module 6600 is
configured to generate an output copy number that reaches or
exceeds the threshold of the sensitivity of an associated detection
module.
[0179] The amplification module 6600 includes a flow member 6610
and a heater 6630. The flow member 6610 can be any suitable flow
member that defines a volume or a series of volumes within which
the prepared solution S3 can flow and/or be maintained to amplify
the target nucleic acid molecules within the solution S3. The
heater 6630 can be any suitable heater or group of heaters coupled
to the flow member 6610 that can heat the prepared solution S3
within the flow member 6610 to perform any of the amplification
operations as described herein. For example, in some embodiments,
the amplification module 6600 (or any of the amplification modules
described herein) can be similar to the amplification modules shown
and described in U.S. Patent Application No. 65/494,145, entitled
"Printed Circuit Board Heater for an Amplification Module," which
is incorporated herein by reference in its entirety.
[0180] In some embodiments, the flow member 6610 defines a single
volume within which the prepared solution S3 is maintained and
heated to amplify the nucleic acid molecules within the prepared
solution S3. In other embodiments, the flow member 6610 can define
a "switchback" or serpentine flow path through which the prepared
solution S3 flows. Similarly stated, the flow member 6610 defines a
flow path that is curved such that the flow path 6618 intersects
the heater 6630 at multiple locations. In this manner, the
amplification module 6600 can perform a "flow through" PCR where
the prepared solution S3 flows through multiple different
temperature regions.
[0181] The flow member 6610 (and any of the flow members described
herein) can be constructed from any suitable material and can have
any suitable dimensions to facilitate the desired amplification
performance for the desired volume of sample. For example, in some
embodiments, the amplification module 6600 (and any of the
amplification modules described herein) can perform 6000.times. or
greater amplification in a time of less than 65 minutes. For
example, in some embodiments, the flow member 6610 (and any of the
flow members described herein) is constructed from at least one of
a cyclic olefin copolymer or a graphite-based material. Such
materials facilitate the desired heat transfer properties into the
flow path 6620. Moreover, in some embodiments, the flow member 6610
(and any of the flow members described herein) can have a thickness
of less than about 0.5 mm. In some embodiments, the flow member
6610 (and any of the flow members described herein) can have a
volume about 150 microliters or greater, and the flow can be such
that at least 10 microliters of sample is amplified. In other
embodiments, at least 20 microliters of sample are amplified by the
methods and devices described herein. In other embodiments, at
least 30 microliters of sample are amplified by the methods and
devices described herein. In yet other embodiments, at least 50
microliters of sample are amplified by the methods and devices
described herein.
[0182] The heater 6630 can be any suitable heater or collection of
heaters that can perform the functions described herein to amplify
the prepared solution S3. In some embodiments, the heater 6630 can
establish multiple temperature zones through which th